DNA-Cu(II) poly(amine) complex membrane as novel catalytic layer for highly sensitive amperometric determination of hydrogen peroxide

A novel hydrogen peroxide biosensor was fabricated by using a DNA-Cu(II) complex as a novel electrocatalyst for the reduction of hydrogen peroxide (H2O2). A polyion complex (PIC) membrane composed of DNA and poly(allylamine) (PAA) functioned as a support matrix for immobilization of electrocatalytic element-copper ion. The circular dichroism (CD) spectrum of the DNA-Cu(II)/PAA membrane in wet state showed that the DNA exists in B-like form within the membrane. Electrochemical measurements of the DNA-Cu(II)/PAA membrane-modified glassy carbon (GC) electrode revealed that the copper ion embedded in the DNA/PAA layer exhibits good electrochemical behaviors, and the electrochemical rate constant between the immobilized copper ion and the GC electrode surface was estimated to be 26.4 s(-1). The resulting DNA-Cu(II)/PAA/GC electrode showed an excellent electrocatalytic activity for the H2O2 reduction. The sensitivity of the sensor for the determination of H2O2 was affected by the amount of each component, such as copper ion, DNA and PAA in the DNA-Cu(II)/PAA membrane. Effects of applied potential, pH, temperature, ionic strength and buffer concentrations upon the response currents of the sensor were also investigated for an optimum analytical performance. Even in the presence of dissolved oxygen, the sensor exhibited highly sensitive and rapid (response time, less than 5 s) response to H2O2. The steady-state cathodic current responses of the sensor obtained at -0.2 V versus Ag/AgCl in air-saturated 50 mM phosphate buffer (pH 5.0) increased linearly up to 135 microM with the detection limit of 50 nM. Interference by ascorbic acid and uric acid due to the reduction of Cu(II) was effectively cancelled by further modification of outermost layer of polyion complex film. In addition, the sensor exhibited good reproducibility and stability.     

Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E_o^’) of −0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 μM with a detection limit of 0.14 μM at a signal/noise ratio of 3. The apparent Michaelis constant () for the electrocatalytic reaction was 22.6 μM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors.     

Direct electrochemistry and electrocatalytic activity of catalase immobilized onto electrodeposited nano-scale islands of nickel oxide

Cyclic voltammetry was used for simultaneous formation and immobilization of nickel oxide nano-scale islands and catalase on glassy carbon electrode. Electrodeposited nickel oxide may be a promising material for enzyme immobilization owing to its high biocompatibility and large surface. The catalase films assembled on nickel oxide exhibited a pair of well defined, stable and nearly reversible CV peaks at about -0.05 V vs. SCE at pH 7, characteristic of the heme Fe (III)/Fe (II) redox couple. The formal potential of catalase in nickel oxide film were linearly varied in the range 1-12 with slope of 58.426 mV/pH, indicating that the electron transfer is accompanied by single proton transportation. The electron transfer between catalase and electrode surface, (k(s)) of 3.7(+/-0.1) s(-1) was greatly facilitated in the microenvironment of nickel oxide film. The electrocatalytic reduction of hydrogen peroxide at glassy carbon electrode modified with nickel oxide nano-scale islands and catalase enzyme has been studied. The embedded catalase in NiO nanoparticles showed excellent electrocatalytic activity toward hydrogen peroxide reduction. Also the modified rotating disk electrode shows good analytical performance for amperometric determination of hydrogen peroxide. The resultant catalase/nickel oxide modified glassy carbon electrodes exhibited fast amperometric response (within 2 s) to hydrogen peroxide reduction (with a linear range from 1 microM to 1 mM), excellent stability, long term life and good reproducibility. The apparent Michaelis-Menten constant is calculated to be 0.96(+/-0.05)mM, which shows a large catalytic activity of catalase in the nickel oxide film toward hydrogen peroxide. The excellent electrochemical reversibility of redox couple, high stability, technical simplicity, lake of need for mediators and short preparations times are advantages of this electrode. Finally the activity of biosensor for nitrite reduction was also investigated.     

Nanomolar detection of hydrogen peroxide at a new polynuclear cluster of tin pentacyanonitrosylferrate nanoparticle-modified carbon ceramic electrode

This work describes a new electrochemical sensor for hydrogen peroxide based on tin pentacyanonitrosylferrate (SnPCNF)-modified carbon ceramic electrode (CCE). The modified electrode was constructed by using a sol-gel technique involving two steps: construction of CCE containing metallic tin (Sn) powder and then electrochemical creation of SnPCNF film on the surface of CCE. The modified electrode was characterized by energy-dispersive X-ray, Fourier transform infrared, scanning electron microscopy, and cyclic voltammetry (CV) techniques. The charge transfer coefficient (α) and charge transfer rate constant (k_s) for the modifying film were calculated. The electrocatalytic activity of the modified electrode toward the reduction of hydrogen peroxide was studied by CV and chronoamperometry. A linear calibration curve was obtained over the hydrogen peroxide concentration range of 0.5 to 69.4 μM using a hydrodynamic amperometric technique. The limit of detection (for a signal-to-noise ratio of 3) and sensitivity were found to be 92 nM and 0.89 μA/μM, respectively. Furthermore, the diffusion coefficient of hydrogen peroxide (D) and catalytic rate constant (k_cat) were calculated.     

Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA

The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.     

A novel electrochemical sensor surface for the detection of hydrogen peroxide using cyclic bisureas/gold nanoparticle composite

A novel electrochemical sensor surface with enhanced sensitivity for the detection of hydrogen peroxide has been developed based on the layer-by-layer assembly of mercapto propionic acid (MPA), cystine-based polymethylene-bridged cyclic bisureas (CBU)/gold nanoparticle (AuNP) and horseradish peroxidase (HRP) on gold electrode. Possibility of a large number of hydrogen bonds, allowed by the chemical and sterical structure of the CBU ensures the proper immobilization of the enzyme in favorable orientation and retention of enzymatic activity. Efficient electron tunneling property of AuNP together with its electrocatalytic activity leads to higher sensitivity in the detection of H(2)O(2). In cyclic voltammetry measurements a cathodic current due to direct electron transfer of HRP is observed which, indicates excellent electrocatalytic activity of the sensor surface. The biosensor surface modified with gold nanoparticle and CBU showed a lower detection limit of 50 nM for hydrogen peroxide. Chronoamperometry is performed at -0.3 V and Michaelis-Menten constant K(M)(app) value is estimated to be 4.5 μM. The newly developed sensor surface showed very high stability, reproducibility and high sensitivity.     

Role of iron in adriamycin biochemistry

Adriamycin forms a chelate with Fe(III) that exhibits complex redox chemistry. The drug ligand is able to directly reduce the bound Fe(III) with the concomitant production of a one-electron oxidized drug radical. This Fe(II) can reduce oxygen to hydrogen peroxide and cleave the peroxide to yield the hydroxyl radical. In addition, the drug X Fe complex can catalyze the transfer of electrons from reduced glutathione to molecular oxygen to yield superoxide, hydrogen peroxide, and hydroxyl radicals. The adriamycin X Fe complex binds to DNA to form a ternary drug X Fe X DNA complex, which is also able to catalyze the thiol-dependent reduction of oxygen and the formation of hydroxyl radical from hydrogen peroxide. As a consequence of this chemistry, the adriamycin X Fe complex can cleave DNA on the addition of glutathione or hydrogen peroxide. Although less well defined, the adriamycin X Fe complex can bind to cell membranes and cause oxidative destruction of these membranes in the presence of thiols or hydrogen peroxide.     

Au nanospheres and nanorods for enzyme-free electrochemical biosensor applications

Au nanocrystals with different morphologies were prepared and used for enzyme-free electrochemical biosensor applications. To investigate the electrocatalytic properties of Au nanocrystals as a function on their morphologies, Au nanocrystals, Au nanospheres (NSs) on silica, Au NSs, and Au nanorods (NRs) with aspect ratios of 1:3 and 1:5, were coated on the screen printed electrodes and further measure the amperometric responses to hydrogen peroxide via three-electrode system. The electrodes modified with Au nanocrystals showed biosensing properties without any enzyme being attached or immobilized at their surface. The hydrogen peroxide detection limits of the biosensors with Au NSs, Au NRs (1:3), and Au NRs (1:5) were 6.48, 8.65, and 9.38 μM (S/N = 3), respectively. The biosensors with Au NSs, Au NRs (1:3), and Au NRs (1:5) showed the sensitivities of 11.13, 54.53, and 58.51 μA/mM, respectively. These results indicate that morphologies of Au nanocrystals significantly influence the sensitivity of the biosensors. In addition, the enzyme-free biosensors with Au nanocrystals were stable for 2 months. Au nanocrystal-based enzyme-free system, which is proposed in this study, can be used as a platform for various electrochemical biosensors.     

Electrocatalytic reduction of protons and oxygen at glassy carbon electrodes coated with a cobalt(II)/platinum(II) porphyrin

The electrocatalytic reduction of protons in 1.0 M perchloric acid at glassy carbon electrodes anodically modified with a Co(II)/Pt(II) porphyrin show shifts of 400 mV versus Ag/AgCl when compared to the same electrodes which have not been anodically modified. Anodic cycling of glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin in this study form stable electroactive films capable of improving both electroreduction of protons to hydrogen and oxygen to both peroxide and water. Electrooxidation of glassy carbon electrodes coated with the free base porphyrin show no improvement in catalytic ability for the reduction of protons in acidic solution or the reduction of molecular oxygen in basic solution. Glassy carbon electrodes coated with the Co(II)/Pt(II) porphyrin indicate, by rotating disk electrochemistry, that the electrocatalysis of oxygen is a two electron process leading to the formation of hydrogen peroxide. Koutecky-Levich plots of the data obtained from the reduction of oxygen at electrode surfaces coated with the Co(II)/Pt(II) porphyrin after oxidation of the surface indicate that 25% of the oxygen is reduced by four electrons directly to water while 75% of the oxygen is reduced by two electrons to hydrogen peroxide.     

Nano polyurethane-assisted ultrasensitive biodetection of H(2)O(2) over immobilized microperoxidase-11

Nanostructured polyurethane (PU) synthesized by an emulsion polymerization with narrow size distribution was employed for the first time directly as a novel matrix for enzyme immobilization to develop sensitively amperometric biosensors. When Microperoxidase-11 (MP-11) was selected as a model protein, the resulting hydrogen peroxide (H(2)O(2)) biosensor exhibited improved sensitivity of 29.6μAmM(-1)cm(-2) with quite good response time of (1.3±0.4)s and remarkable limit of detection as low as 10pM (S/N 3) over existing protocols. A linear calibration curve for hydrogen peroxide was obtained up to 1.3μM under the optimized conditions with a relative low calculated Michaelis-Menten constant (K(M)(app)) (1.87±0.05)μM, which indicated the enhanced enzymatic affinity of MP-11 to H(2)O(2) via PU. The possible interferents had negligible effect on the response current and time of the prepared biosensor. Results suggest that the PU nanoparticles (PU-NPs) with good biocompatibility and sufficient interfacial adhesion hold promise as an attractive support material for construction of ultrasensitive amperometric biosensor.     

Interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX, a solid state spectroscopy study

To investigate the nature of binding of quinoline antimalarial drugs to heme and to extract experimental evidence for this binding, the interaction of ferriprotoporphyrin IX (FP) with chloroquine and quinacrine (both of which have a similar side chain) and quinoline methanol antimalarials quinine and mefloquine has been studied using IR and NIR-Raman spectroscopy in the solid state. Attenuated total reflectance infrared spectroscopic data clearly show that heme in chloroquine-FP complex is not μ-oxo dimeric indicating that the hypothesis that chloroquine binds to FP μ-oxo dimer with a stoichiometry of 1 chloroquine:2 μ-oxo dimers is not valid in the solid state. Moreover, the first vibrational spectroscopy evidence is presented for the formation of hydrogen bonding between a propionate group of heme and the tertiary amino nitrogen of chloroquine and quinacrine. Raman spectroscopy data does not provide any evidence to support the formation of a similar salt bridge in the complexes of FP with quinine and mefloquine; however, it suggests that the interaction of these drugs with FP happens through coordination of the Fe(III) center of the porphyrin to the 9-hydroxy group of the drug.     

Interaction of heliquinomycin with single-stranded DNA inhibits MCM4/6/7 helicase

The antibiotic heliquinomycin inhibited cellular DNA replication at IC(50) of 2.5 μM without affecting level of chromatin-bound MCM4 and without activating the DNA replication stress checkpoint system, suggesting that heliquinomycin perturbs DNA replication mainly by inhibiting the activity of replicative DNA helicase that unwinds DNA duplex at replication forks. Among the DNA helicases involved in DNA replication, DNA helicase B was inhibited by heliquinomycin at IC(50) of 4.3 μM and RECQL4 helicase at IC(50) of 14 μM; these values are higher than that of MCM4/6/7 helicase (2.5 μM). These results suggest that heliquinomycin mainly targets actions of the replicative DNA helicases. Gel-retardation experiment indicates that heliquinomycin binds to single-stranded DNA. The single-stranded DNA-binding ability of MCM4/6/7 was affected in the presence of heliquinomycin. The data suggest that heliquinomycin inhibits the DNA helicase activity of MCM4/6/7 complex by stabilizing its interaction with single-stranded DNA.     

Mithramycin forms a stable dimeric complex by chelating with Fe(II): DNA-interacting characteristics, cellular permeation and cytotoxicity

Mith (mithramycin) forms a 2:1 stoichiometry drug–metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding affinity between Mith and Fe(II) is much greater than other divalent metal ions, including Mg(II), Zn(II), Co(II), Ni(II) and Mn(II). The [(Mith)₂–Fe(II)] complex binds to DNA and induces a conformational change of DNA. Kinetic analysis of surface plasmon resonance studies revealed that the [(Mith)₂–Fe(II)] complex binds to DNA duplex with higher affinity compared with the [(Mith)₂–Mg(II)] complex. A molecular model of the Mith-DNA–Metal(II) complex is presented. DNA-break assay showed that the [(Mith)₂–Fe(II)] complex was capable of promoting the one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide. Intracellular Fe(II) assays and fluorescence microscopy studies using K562 indicated that this dimer complex maintains its structural integrity and permeates into the inside of K562 cells, respectively. The [(Mith)₂–Fe(II)] complex exhibited higher cytotoxicity than the drug alone in some cancer cell lines, probably related to its higher DNA-binding and cleavage activity. Evidences obtained in this study suggest that the biological effects caused by the [(Mith)₂–Fe(II)] complex may be further explored in the future.     

Single-stranded DNA modification by an oligonucleotide-phthalocyanine Fe(II) conjugate: the kinetic features and the process mechanism

Catalytic oxidative modification of a single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like the earlier studied oligonucleotide conjugates containing metallocomplexes tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic features and optimal regimes of DNA oxidation with hydrogen peroxide were found.     

A bienzyme channeling glucose sensor with a wide concentration range based on co-entrapment of enzymes in SBA-15 mesopores

A novel bienzyme-channeling sensor was constructed by entrapping glucose oxidase (GOD) and horseradish peroxidase (HRP) in the mesopores of well-ordered hexagonal mesoporous silica structures (SBA-15). The SBA-15 mesoporous materials accelerated the electron transfer between the entrapped HRP and electrode. Both HRP and GOD retained their catalytic activities in the bienzyme-entrapped SBA-15 film. In presence of glucose the enzymatic reaction of GOD-glucose-dissolved oxygen system generated hydrogen peroxide in the bienzyme-entrapped mesopores, which was immediately reduced at -0.40 V by an electrocatalytic reaction with the HRP entrapped in the same mesopore to lead to a sensitive and fast amperometric response. Thus the bienzyme channeling could be used for the detection of glucose with excellent performance without the addition of any mediator. Optimization of the experimental parameters was performed with regard to pH, operating potential and temperature. The detection limit was down to 2.7 x 10(-7)M with a very wide linear range from 3.0 x 10(-6) to 3.4 x 10(-2)M. The constructed bienzyme channeling provided a strategy for amperometric detection of oxidase substrates by co-entrapping the corresponding oxidase and HRP in the mesoporous materials.     

Reductive determination of hydrogen peroxide with MWCNTs-Pd nanoparticles on a modified glassy carbon electrode

This paper introduces the use of multi walled carbon nanotubes (MWCNTs) with palladium (Pd) nanoparticles in the electrocatalytic reduction of hydrogen peroxide (H(2)O(2)). We have developed and characterized a biosensor for H(2)O(2) based on Nafion(?) coated MWCNTs-Pd nanoparticles on a glassy carbon electrode (GCE). The Nafion(?)/MWCNTs-Pd/GCE electrode was easily prepared in a rapid and simple procedure, and its application improves sensitive determination of H(2)O(2). Characterization of the MWCNTs-Pd nanoparticle film was performed with transmission electron microscopy (TEM), Raman, and X-ray photoelectron spectroscopy (XPS). Cyclic voltammetry (CV) and amperometry (at an applied potential of -0.2V) measurements were used to study and optimize performance of the resulting peroxide biosensor. The proposed H(2)O(2) biosensor exhibited a wide linear range from 1.0 μM to 10 mM and a low detection limit of 0.3 μM (S/N=3), with a fast response time within 10s. Therefore, this biosensor could be a good candidate for H(2)O(2) analysis.     

Oxidative DNA cleavage mediated by a new copper (II) terpyridine complex: crystal structure and DNA binding studies

The copper (II) complex [Cu(Itpy)(2)](ClO(4))(2) (1), (Itpy=imidazole terpyridine) has been synthesized and structurally characterized. Crystal structure of the complex shows the complex to be a monomeric copper (II) species with two Itpy ligands coordinated to the metal ion to give a six coordinate complex. The complex has a distorted octahedral geometry with axial elongation. Variable temperature crystal structure data shows dynamic nature of the Jahn-Teller distortion. The complex is an avid DNA binder with a binding constant of 4.26+/-0.20x10(3)M(-1). Observed changes in the viscosity and circular dichroic spectrum of calf thymus DNA solution in the presence of complex 1 suggests intercalative binding of complex 1 to DNA. The complex cleaves supercoiled pBR322 DNA oxidatively in the presence of hydrogen peroxide.     

Layer-by-layer self-assembly of functionalized graphene nanoplates for glucose sensing in vivo integrated with on-line microdialysis system

In this work, a novel amperometric biosensor for hydrogen peroxide was fabricated through the layer-by-layer (LBL) self-assembling of amine-terminated ionic liquid (IL-NH(2)), and sulfonic acid (SO(3)(-)) functionalized graphene by covalent bonding. The modification of the two functionalities introduced positive and negative charge onto the surface of graphene respectively, thus facilitating the formation of a multilayer film denoted with {IL-RGO/S-RGO}(n) through electrostatic interaction and further immobilization of glucose oxidase (GOx). The resulting {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor displayed an excellent response to glucose at a potential of -200 mV. Combined with on-line microdialysis system, the glucose biosensor in the on-line system showed good linear range from 10 μM to 500 μM with the detection limit of 3.33 μM (S/N=3). Consequently, the basal level of glucose in the striatum of anesthetic rats was calculated to be 0.376 ± 0.028 mM (mean ± s.d., n=3). The {IL-RGO/S-RGO}(n)/GOx/Nafion biosensor was further applied for in vivo sensing of the glucose level in the striatum when rats received intraperitoneal (i.p.) injection of 30 μL insulin, which resulted in an obvious decrease in the extracellular concentration of glucose within 30 min. The method was proved to be sensitive and reproducible, which enabled its promising application in physiology and pathology.     

Electrochemical study of the effect of nano-zinc oxide on microperoxidase and its application to more sensitive hydrogen peroxide biosensor preparation

Direct electron transfer reactions of microperoxidase were achieved with the help of semiconductive zinc oxide nanoparticles on a pyrolytic graphite electrode. The enzyme could also exhibit fine electrocatalytic activity towards the reduction of hydrogen peroxide. Thereby, a hydrogen peroxide biosensor was constructed based on the electrocatalysis of microperoxidase. Further studies revealed that after irradiating the microperoxidase/zinc oxide nanoparticles co-modified electrode with UV light for 4h, the catalytic ability of microperoxidase could be greatly promoted, which could be beneficial to developing more sensitive hydrogen peroxide biosensors. As comparison, it was found that the catalytic activity of the enzyme would be depressed if microperoxidase/agarose co-modified electrode was irradiated. We supposed it was the photovoltaic effect of the zinc oxide nanoparticles that improved the catalytic ability of microperoxidase.     

Structural organization of copper(II) ions in complexes with DNA

The interaction of Cu(II) ions with native and denatured DNA as a function of ionic strength of the solution was studied by the equilibrium dialysis method. Graphical analysis of binding isotherms confirmed the occurrence of interstrand and intrastrand binding of Cu(II) with DNA and made possible determination of the respective binding constants. To facilitate interpretation of the data, a new molecular model of Cu(II)-DNA binding has been proposed, assuming interstrand intercalation of one Cu(II) ion between two GC pairs both in the successive even and odd groups of GC pairs, and interstrand binding of Cu(II) to the isolated GC pairs, with the exception of T-C-T and T-G-T sequences. In agreement with this model, the DNA-Cu(II) complex is most stable under the equilibrium with free Cu(II) ions at 4 degrees C, pH 6 when the molar ratio of GC pairs to Cu(II) ions bound interstrandially attains GC/Cuinter = 2 +/- 0.1.