Electrical Stimuli-Responsive Decomposition of Layer-by-Layer Films Composed of Polycations and TEMPO-Modified Poly(acrylic acid).

We previously reported that layer-by-layer (LbL) film prepared by a combination of 2,2,6,6-tetramethylpiperidinyl N-oxyl (TEMPO)-modified polyacrylic acid (PAA) and polyethyleneimine (PEI) were decomposed by application of an electric potential. However, there have been no reports yet for other polycationic species. In this study, LbL films were prepared by combining various polycationics (PEI, poly(allylamine hydrochloride) (PAH), poly(diallydimethylammonium chloride) (PDDA), and polyamidoamine (PAMAM) dendrimer) and TEMPO-PAA, and the decomposition of the thin films was evaluated using cyclic voltammetry (CV) and constant potential using an electrochemical quartz crystal microbalance (eQCM). When a potential was applied to an electrode coated on an LbL thin film of polycations and TEMPO-PAA, an oxidation potential peak (Epa) was obtained around +0.6 V vs. Ag/AgCl in CV measurements. EQCM measurements showed the decomposition of the LbL films at voltages near the Epa of the TEMPO residues. Decomposition rate was 82% for the (PEI/TEMPO-PAA)5 film, 52% for the (PAH/TEMPO-PAA)5 film, and 49% for the (PDDA/TEMPO-PAA)5 film. It is considered that the oxoammonium ion has a positive charge, and the LbL films were decomposed due to electrostatic repulsion with the polycations (PEI, PAH, and PDDA). These LbL films may lead to applications in drug release by electrical stimulation. On the other hand, the CV of the (PAMAM/TEMPO-PAA)5 film did not decompose. It is possible that the decomposition of the thin film is not promoted, probably because the amount of TEMPO-PAA absorbed is small.

Effect of cytosine-Ag+-cytosine base pairing on the redox potential of the Ag+/Ag couple and the chemical reduction of Ag+ to Ag by tetrathiafulvalene.

The redox properties of metallo-base pairs remain to be elucidated. Herein, we report the detailed 1H/13C/109Ag NMR spectroscopic and cyclic voltammetric characterisation of the [Ag(cytidine)2]+ complex as isolated cytosine-Ag+-cytosine (C-Ag+-C) base pairs. We also performed comparative studies between cytidine/Ag+ and other nucleoside/Ag+ systems by using cyclic voltammetry measurements. In addition, to evaluate the effect of [Ag(cytidine)2]+ formation on the chemical reduction of Ag+ to Ag, we utilised the redox reaction between Ag+ and tetrathiafulvalene (TTF). We found that Ag+-mediated base pairing lowers the redox potential of the Ag+/Ag couple. In addition, C-Ag+-C base pairing makes it more difficult to reduce captured Ag+ ions than in other nucleoside/Ag+ systems. Remarkably, the cytidine/Ag+ system can be utilised to control the redox potential of the Ag+/Ag couple in DMSO. This feature of the cytidine/Ag+ system may be exploited for Ag nanoparticle synthesis by using the redox reaction between Ag+ and TTF.

Chemical reduction of Ag+ to Ag employing organic electron donors: evaluation of the effect of Ag+-mediated cytosine-cytosine base pairing on the aggregation of Ag nanoparticles.

Ag+-mediated base pairing is valuable for synthesising DNA-based silver nanoparticles (AgNPs) and nanoclusters (AgNCs). Recently, we reported the formation of a [Ag(cytidine)2]+ complex in dimethyl sulfoxide (DMSO), which facilitated the evaluation of the effect of cytosine-Ag+-cytosine (C-Ag+-C) base pairing on the degree of AgNP aggregation in solution. As an aprotic solvent, DMSO was expected to dissolve the [Ag(cytidine)2]+ complex, and powerful reducing agents, such as organic electron donors. In this study, the chemical reduction of a cytidine/Ag+ system using a powerful reducing agent tetrakis(dimethylamino)ethylene (TDAE) was investigated. 1H/13C/15N NMR spectroscopic evidence was obtained to identify the iminium dication (TDAE2+), which is an oxidised form of TDAE. The results were compared with those obtained using another organic electron donor, tetrathiafulvalene (TTF), which exhibits a relatively lower reduction activity than TDAE. AgNPs prepared via redox reaction between [Ag(cytidine)2]+ and organic electron donors (TDAE and TTF) were characterised using UV-Vis spectroscopy and nanoparticle tracking analysis. It was found that the formation of C-Ag+-C base pairing inhibited the aggregation of AgNPs in solution. In addition, in the presence of cytidine, the total concentration of the AgNP solution was affected by the reduction activity of the reducing agent.

Catalysis of electro-oxidation of antibiotics by nitroxyl radicals and the electrochemical sensing of vancomycin.

Quantifying drug concentrations in vivo quickly and easily is possible using electrochemical methods. The present study describes the electrochemical detection of vancomycin (VCM) and other antibiotics from the current obtained using nitroxyl radicals as electrocatalysts. Nortropine N-oxyl (NNO), which is more active than 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), a typical nitroxyl radical compound, produced greater current values for drugs with intramolecular hydroxy groups and secondary and tertiary amines. However, because the catalytic action of NNO is inactivated by primary amines in the substrate, VCM and teicoplanin with primary amines could not be detected. TEMPO was less active than NNO but not inactivated against primary amines. Therefore, electrochemical sensing of vancomycin was done using 4-acetamido-2,2,6,6-tetramethylpiperidine 1-oxyl (A-TEMPO), which has a greater oxidation capacity than TEMPO due to its electron-withdrawing groups. As a result, the current of A-TEMPO increased in the low concentration range of VCM as compared to TEMPO. This method also was able to quantify VCM in the concentration range of 10-100 µM, which is an important concentration range for drug monitoring in blood.

Adsorption and Release of Rose Bengal on Layer-by-Layer Films of Poly(Vinyl Alcohol) and Poly(Amidoamine) Dendrimers Bearing 4-Carboxyphenylboronic Acid.

Phenylboronic acid-bearing polyamidoamine dendrimer (PBA-PAMAM)/poly(vinyl alcohol) (PVA) multilayer films were prepared through the layer-by-layer (LbL) deposition of PBA-PAMAM solution and PVA solution. PBA-PAMAM/PVA films were constructed successfully through the formation of boronate ester bonds between the boronic acid moiety in PBA and 1,3-diol units in PVA. When the (PBA-PAMAM/PVA)5 films were immersed in rose bengal (RB) solution, RB was adsorbed onto the LbL films. The amount of RB adsorbed was higher in the LbL films immersed in acidic solution than in basic solution. The release of RB from the LbL films was also promoted in the basic solution, while it was suppressed in the acidic solution. The boronic acid ester is oxidized to phenol by hydrogen peroxide (H2O2) and the carbon-boron bond is cleaved, so that the (PBA-PAMAM/PVA)5 films can be decomposed by immersion in H2O2 solution. Therefore, when RB-adsorbed (PBA-PAMAM/PVA)5 films were immersed in H2O2 solution, the release of RB was moderately promoted when the solution was weakly acidic.

Decomposition of Glucose-Sensitive Layer-by-Layer Films Using Hemin, DNA, and Glucose Oxidase.

Glucose-sensitive films were prepared through the layer-by-layer (LbL) deposition of hemin-modified poly(ethyleneimine) (H-PEI) solution and DNA solution (containing glucose oxidase (GOx)). H-PEI/DNA + GOx multilayer films were constructed using electrostatic interactions. The (H-PEI/DNA + GOx)5 film was then partially decomposed by hydrogen peroxide (H2O2). The mechanism for the decomposition of the LbL film was considered to involve more reactive oxygen species (ROS) that were formed by the reaction of hemin and H2O2, which then caused nonspecific DNA cleavage. In addition, GOx present in the LbL films reacts with glucose to generate hydrogen peroxide. Therefore, decomposition of the (H-PEI/DNA + GOx)5 film was observed when the thin film was immersed in a glucose solution. (H-PEI/DNA + GOx)5 films exposed to a glucose solution for periods of 24, 48 72, and 96 h indicated that the decomposition of the film increased with the time to 9.97%, 16.3%, 23.1%, and 30.5%, respectively. The rate of LbL film decomposition increased with the glucose concentration. At pH and ionic strengths close to physiological conditions, it was possible to slowly decompose the LbL film at low glucose concentrations of 1-10 mM.

Crystal structure of a DNA duplex cross-linked by 6-thioguanine-6-thioguanine disulfides: reversible formation and cleavage catalyzed by Cu(ii) ions and glutathione.

Herein, we determined the crystal structure of a DNA duplex containing consecutive 6-thioguanine-6-thioguanine disulfides. The disulfide bonds were reversibly formed and cleaved in the presence of Cu(ii) ions and glutathione. To our knowledge, this is the first reaction in which metal ions efficiently accelerated disulfide bond formation between thio-bases in duplexes.

Preparation of Hydrogen Peroxide Sensitive Nanofilms by a Layer-by-Layer Technique.

H2O2-sensitive nanofilms composed of DNA and hemin-appended poly(ethyleneimine) (H-PEI) were prepared by a layer-by-layer deposition of DNA and H-PEI through an electrostatic interaction. The (H-PEI/DNA)5 film was decomposed by addition of 10 mM H2O2. H2O2-induced decomposition was also confirmed in the hemin-containing (PEI/DNA)5 in which hemin molecules were adsorbed by a noncovalent bond to the nanofilm. On the other hand, the (PEI/DNA)5 film containing no hemin and the (H-PEI/PSS)5 film using PSS instead of DNA did not decompose even with 100 mM H2O2. The mechanism of nanofilm decomposition was thought that more reactive oxygen species (ROS) was formed by reaction of hemin and H2O2 and then the ROS caused DNA cleavage. As a result (H-PEI/DNA)5 and hemin-containing (PEI/DNA)5 films were decomposed. The decomposition rate of these nanofilms were depended on concentration of H2O2, modification ratio of hemin, pH, and ionic strength.

Preparation of Microparticles Capable of Glucose-Induced Insulin Release under Physiological Conditions.

Hydrogen peroxide (H2O2)-sensitive layer-by-layer films were prepared based on combining phenyl boronic acid (PBA)-modified poly(allylamine) (PAH) with shikimic acid (SA)-modified-PAH through boronate ester bonds. These PBA-PAH/SA-PAH multilayer films could be prepared in aqueous solutions at pH 7.4 and 9.0 in the presence of NaCl. It is believed that the electrostatic repulsion between the SA-PAH and PBA-PAH was diminished and the formation of ester bonds between the SA and PBA was promoted in the presence of NaCl. These films readily decomposed in the presence of H2O2 because the boronate ester bonds were cleaved by an oxidation reaction. In addition, SA-PAH/PBA-PAH multilayer films combined with glucose oxidase (GOx) were decomposed in the presence of glucose because GOx catalyzes the oxidation of D-glucose to generate H2O2. The surfaces of CaCO3 microparticles were coated with PAH/GOx/(SA-PAH/PBA-PAH)5 films that absorbed insulin. A 1 mg quantity of these particles released up to 10 µg insulin in the presence 10 mM glucose under physiological conditions.

Preparation of Nafion/Polycation Layer-by-Layer Films for Adsorption and Release of Insulin.

Thin films were prepared using layer-by-layer (LbL) deposition of Nafion (NAF) and polycations such as poly(allylamine hydrochloride) (PAH), poly(ethyleneimine) (PEI), and poly(diallydimethylammonium chloride) (PDDA). Insulin was then adsorbed on the NAF-polycation LbL films by immersion in an insulin solution. The NAF-polycation LbL films were characterized using a quartz crystal microbalance and an atomic force microscope. The release of insulin from the LbL films was characterized using UV-visible adsorption spectroscopy and fluorescence emission spectroscopy. The greatest amount of insulin was adsorbed on the NAF-PAH LbL film. The amount of insulin adsorbed on the (NAF/PAH)5NAF LbL films by immersion in a 1 mg mL-1 insulin solution at pH 7.4 was 61.8 µg cm-2. The amount of insulin released from the LbL films was higher when immersed in insulin solutions at pH 2.0 and pH 9.0 than at pH 7.4. Therefore, NAF-polycations could be employed as insulin delivery LbL films under mild conditions and as an insulin release control system according to pH change.

A metallo-DNA nanowire with uninterrupted one-dimensional silver array.

The double-helix structure of DNA, in which complementary strands reversibly hybridize to each other, not only explains how genetic information is stored and replicated, but also has proved very attractive for the development of nanomaterials. The discovery of metal-mediated base pairs has prompted the generation of short metal-DNA hybrid duplexes by a bottom-up approach. Here we describe a metallo-DNA nanowire-whose structure was solved by high-resolution X-ray crystallography-that consists of dodecamer duplexes held together by four different metal-mediated base pairs (the previously observed C-Ag-C, as well as G-Ag-G, G-Ag-C and T-Ag-T) and linked to each other through G overhangs involved in interduplex G-Ag-G. The resulting hybrid nanowires are 2 nm wide with a length of the order of micrometres to millimetres, and hold the silver ions in uninterrupted one-dimensional arrays along the DNA helical axis. The hybrid nanowires are further assembled into three-dimensional lattices by interactions between adenine residues, fully bulged out of the double helix.

Structure Determination of an Ag(I) -Mediated Cytosine-Cytosine Base Pair within DNA Duplex in Solution with (1) H/(15) N/(109) Ag NMR Spectroscopy.

The structure of an Ag(I) -mediated cytosine-cytosine base pair, C-Ag(I) -C, was determined with NMR spectroscopy in solution. The observation of 1-bond (15) N-(109) Ag J-coupling ((1) J((15) N,(109) Ag): 83 and 84 Hz) recorded within the C-Ag(I) -C base pair evidenced the N3-Ag(I) -N3 linkage in C-Ag(I) -C. The triplet resonances of the N4 atoms in C-Ag(I) -C demonstrated that each exocyclic N4 atom exists as an amino group (-NH2 ), and any isomerization and/or N4-Ag(I) bonding can be excluded. The 3D structure of Ag(I) -DNA complex determined with NOEs was classified as a B-form conformation with a notable propeller twist of C-Ag(I) -C (-18.3±3.0°). The (109) Ag NMR chemical shift of C-Ag(I) -C was recorded for cytidine/Ag(I) complex (δ((109) Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C-Ag(I) -C base pair.

Exploring a DNA Sequence for the Three-Dimensional Structure Determination of a Silver(I)-Mediated C-C Base Pair in a DNA Duplex By (1)H NMR Spectroscopy.

Recently, we discovered novel silver(I)-mediated cytosine-cytosine base pair (C-Ag(I)-C) in DNA duplexes. To understand the properties of these base pairs, we searched for a DNA sequence that can be used in NMR structure determination. After extensive sequence optimizations, a non-symmetric 15-base-paired DNA duplex with a single C-Ag(I)-C base pair flanked by 14 A-T base pairs was selected. In spite of its challenging length for NMR measurements (30 independent residues) with small sequence variation, we could assign most non-exchangeable protons (254 out of 270) and imino protons for structure determination.

High-Resolution Crystal Structure of a Silver(I)-RNA Hybrid Duplex Containing Watson-Crick-like C-Silver(I)-C Metallo-Base Pairs.

Metallo-base pairs have been extensively studied for applications in nucleic acid-based nanodevices and genetic code expansion. Metallo-base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo-base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T-Hg(II)-T base pairs. Herein, we have determined a high-resolution crystal structure of the second natural metallo-base pair between pyrimidine bases C-Ag(I)-C formed in an RNA duplex. One Ag(I) occupies the center between two cytosines and forms a C-Ag(I)-C base pair through N3-Ag(I)-N3 linear coordination. The C-Ag(I)-C base pair formation does not disturb the standard A-form conformation of RNA. Since the C-Ag(I)-C base pair is structurally similar to the canonical Watson-Crick base pairs, it can be a useful building block for structure-based design and fabrication of nucleic acid-based nanodevices.

Structures, physicochemical properties, and applications of T-Hg(II)-T, C-Ag(I)-C, and other metallo-base-pairs.

Recently, metal-mediated base-pairs (metallo-base-pairs) have been studied extensively with the aim of exploring novel base-pairs; their structures, physicochemical properties, and applications have been studied. This trend has become more evident after the discovery of Hg(II)-mediated thymine-thymine (T-Hg(II)-T) and Ag(I)-mediated cytosine-cytosine (C-Ag(I)-C) base-pairs. In this article, we focus on the basic science and applications of these metallo-base-pairs, which are composed of natural bases.

Direct detection of the mercury-nitrogen bond in the thymine-Hg(II)-thymine base-pair with (199)Hg NMR spectroscopy.

We have observed the 1-bond (199)Hg-(15)N J-coupling ((1)J((199)Hg,(15)N) = 1050 Hz) within the Hg(II)-mediated thymine-thymine base pair (T-Hg(II)-T). This strikingly large (1)J((199)Hg,(15)N) is the first one for canonical sp(2)-nitrogen atoms, which can be a sensitive structure-probe of N-mercurated compounds and a direct evidence for N-mercuration.

Hg2+-trapping beads: Hg2+-specific recognition through thymine-Hg(II)-thymine base pairing.

Mercury pollution poses a severe threat to human health. To remove Hg(2+) from contaminated water, we synthesized Hg(2+)-trapping beads that include oligo-thymidine functionalities that can form thymine-Hg(II)-thymine base pairs on the solid support. The beads can selectively trap Hg(2+) even in the presence of other metal cations. More interestingly, Hg(2+)-trapping efficiency was higher in the presence of the co-existing cations. Thus, the developed Hg(2+)-trapping beads can capture Hg(2+) without affecting the mineral balance of water so much. The Hg(2+)-trapping beads presented here show promise for removing Hg(2+) from environmental water.

The structure of metallo-DNA with consecutive thymine-HgII-thymine base pairs explains positive entropy for the metallo base pair formation.

We have determined the three-dimensional (3D) structure of DNA duplex that includes tandem Hg(II)-mediated T-T base pairs (thymine-Hg(II)-thymine, T-Hg(II)-T) with NMR spectroscopy in solution. This is the first 3D structure of metallo-DNA (covalently metallated DNA) composed exclusively of 'NATURAL' bases. The T-Hg(II)-T base pairs whose chemical structure was determined with the (15)N NMR spectroscopy were well accommodated in a B-form double helix, mimicking normal Watson-Crick base pairs. The Hg atoms aligned along DNA helical axis were shielded from the bulk water. The complete dehydration of Hg atoms inside DNA explained the positive reaction entropy (ΔS) for the T-Hg(II)-T base pair formation. The positive ΔS value arises owing to the Hg(II) dehydration, which was approved with the 3D structure. The 3D structure explained extraordinary affinity of thymine towards Hg(II) and revealed arrangement of T-Hg(II)-T base pairs in metallo-DNA.

A practical fluorometric assay method to measure lysosomal acid lipase activity in dried blood spots for the screening of cholesteryl ester storage disease and Wolman disease.

Fluorometric measurements of 4-methylumbelliferone (4-MU) are generally used to screen lysosomal storage diseases (LSDs) using dried blood spots (DBSs). However, in DBS, it is difficult to measure lysosomal acid lipase (LAL) activity due to the influence of other lipases in whole blood. Recently, Hamilton used a fluorometric enzyme assay with 4-MU derivatives to measure the LAL activity in DBS. This method requires mercury chloride as stopping reagent, and the fluorescence intensity of 4-MU was measured at an acidic pH. We report a revised method to measure the LAL activity without using toxic mercury chloride and to measure the fluorescence intensity of 4-MU at a basic pH. For this measurement, we established a more practical method that does not require mercury chloride. The LAL activity in DBS was measured in 51 normal controls, seven obligate carriers and seven patients with CESD. The average LAL activities ± SD in the DBS from the normal, obligate carriers and CESD patients were 0.68 ± 0.2 (range: 0.3-1.08), 0.21 ± 0.1 (range: 0.11-0.41) and 0.02 ± 0.02 (range: 0-0.06) nmol/punch/h, respectively. There was a significant difference between the normal and the CESD. Our method does not require toxic mercury chloride and is an appropriate revised enzyme assay using DBS for screening patients with CESD.

Thermodynamic and structural properties of the specific binding between Ag⁺ ion and C:C mismatched base pair in duplex DNA to form C-Ag-C metal-mediated base pair.

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences.