Direct Gas-Phase Derivatization by Employing Tandem µ-Reactor-Gas Chromatography/Mass Spectrometry: Case Study of Trifluoroacetylation of 4,4'-Methylenedianiline.

Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) is a promising technique allowing the rapid characterization of the polymer structure and additives of microgram-scale plastics. However, the Py-GC/MS analysis of polymers with urethane bonds is challenging because they produce highly reactive pyrolyzates such as amines and isocyanates polymerizing in the GC column, which limits the efforts to elucidate the pyrolysis mechanism and plastic characterization by online GC analysis. Herein, a novel pyrolysis-gas-phase derivatization-GC/MS (Py-GPD-GC/MS) technique was developed, allowing the pyrolysis of polymers and the subsequent direct gas-phase derivatization of pyrolyzates, employing a modified tandem µ-reactor-GC/MS system. This work conducted the gas-phase trifluoroacetylation of 4,4'-methylenedianiline (MDA), which is one of the major polyurethane (PU) pyrolyzates, using N-methyl-bis-trifluoroacetamide (MBTFA) as a derivatization agent. The trifluoroacetylation gas-phase reaction was monitored by in situ GC/MS analysis and the effects of derivatization conditions were investigated. The highest MDA conversion observed was 65.6 area %. Furthermore, the sequential PU pyrolysis and direct trifluoroacetylation of PU pyrolyzates in the first µ-reactor and second µ-reactor, respectively, were successfully operated, achieving the inhibited polymerization and detection of trifluoroacetylated derivatives. Thus, the Py-GPD-GC/MS method has a significant potential to be applied for other combinations of pyrolyzates and derivatization reactions, enabling deeper characterization of plastics producing highly reactive pyrolyzates that cannot be accurately analyzed by conventional Py-GC/MS analysis.

Fluorescent Trimethylated Naphthyridine Derivative with an Aminoalkyl Side Chain as the Tightest Non-aminoglycoside Ligand for the Bacterial A-site RNA.

The bacterial ribosomal decoding region of the aminoacyl-tRNA site (A-site) is one of the most validated target RNAs for antibiotic agents. Although natural aminoglycosides are well-characterized A-site binding ligands, high off-target effects and the growing emergence of bacterial resistance against aminoglycosides limit their clinical use. To circumvent these concerns with the aminoglycoside family, non-aminoglycoside A-site binding ligands have great potential as novel antibiotics against bacterial infections. This work describes a new class of small heterocyclic ligands based on the 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) structure for the bacterial (Escherichia coli) A-site. ATMND possessing an aminoethyl side chain is found to strongly and selectively bind to the internal loop of the A-site (Kd =0.44 µm; pH 7.0, I=0.06 m, 5 °C). Significantly, this ligand shows the tightest binding reported to date among non-aminoglycoside ligands. The binding study based on the thermodynamics and molecular modelling reveals key molecular interactions of ATMND-C2 -NH2 for high affinity to the A-site. This ligand is also demonstrated to be applicable to the fluorescence indicator displacement assay for assessing ligand/A-site interactions.

Hydrogenation Reactions during Pyrolysis-Gas Chromatography/Mass Spectrometry Analysis of Polymer Samples Using Hydrogen Carrier Gas.

Pyrolysis-gas chromatography/mass spectrometry of polymer samples is studied focusing on the effect of hydrogen (H2) carrier gas on chromatographic and spectral data. The pyrograms and the related mass spectra of high density polyethylene (HDPE), low density polyethylene, and polystyrene (PS) serve to illustrate the differences between the species formed in H2 and the helium environment. Differences in the pyrograms and the spectra are generally thought to be a result of the hydrogenation reaction of the pyrolyzates. From the peak intensity changes in the pyrograms of HDPE and PS, hydrogenation of unsaturated pyrolyzates is concluded to occur when the pyrolysis is done in H2. Moreover, additional hydrogenation of the pyrolyzates occurs in the electron ionization source of a MS detector when H2 is used as a carrier gas. Finally, the applicability of mass spectral libraries to characterize pyrograms obtained in H2 is illustrated using 24 polymers. The effect of the hydrogenation reaction on the library search results is found to be negligible for most polymer samples with polar and nonpolar monomer units.

Electrochemical enzymatic biosensor with long-term stability using hybrid mesoporous membrane.

An acetylcholinesterase-immobilized sensor unit was successfully prepared by encapsulating the enzyme within hybrid mesoporous silica membranes (F127-MST). Through a novel combination with tetracyanoquinodimethane, both acetylcholine and organophosphorus pesticides were successfully detected with high sensitivity. Furthermore, we manufactured the working prototype of an enzyme sensor with this sensor unit for detecting dichlorvos, aldicarb and parathion. At present, the detection limit in this working prototype either equaled or surpassed that of others. Also, we have the advantage of increased stability of the enzyme against the outer environment by encapsulation of the enzymes into a silica nanospace. Consequently, acetylcholinesterase immobilized in F127-MST is a practical sensor with high sensitivity, reusability, and storage stability.

The effect of LNA nucleobases as enhancers for the binding of amiloride to an abasic site in DNA/DNA and DNA/RNA duplexes.

We report on a significant effect of locked nucleic acid (LNA) nucleobases on the binding of amiloride for abasic site (AP)-containing DNA duplexes. Fluorescence titration experiments showed that the binding affinity of amiloride for the target thymine (T) opposite an AP site significantly improves for the DNA duplexes possessing LNA nucleobases that flank the AP site, compared to the corresponding normal DNA duplexes. In particular, LNA flanking nucleobases on both 5'- and 3'-sides of the AP site are found to be effective for the enhancement of the binding affinity. From thermodynamic characterization of the amiloride binding, the loss in the binding entropy is remarkably reduced for the LNA-containing DNA duplexes, which is indeed responsible for the enhanced affinity of amiloride. Moreover, such an effect of LNA nucleobases was also observed for amiloride binding to DNA/RNA hybrid duplexes.

Quantitative assessment of nano-plastic aerosol particles emitted during machining of carbon fiber reinforced plastic.

Focusing on the relatively unexplored presence of micro- and nano-plastic aerosol particles, this study quantitatively assessed the emission of nano-plastic particles during the machining of carbon fiber reinforced plastic (CFRP) in the working environment. Measurements of aerosol particles smaller than 1 µm in size were performed by aerosol mass spectrometry. The findings revealed that concentrations of carbonous aerosol particles (organic aerosol and refractory black carbon (rBC)) were higher during working hours than during non-working hours. Positive matrix factorization identified CFRP particles as a significant source, contributing an average of approximately 30% of concentration of carbonous aerosol particles during working hours. This source apportionment was corroborated by the presence of bisphenol A and F fragments, principal components of the epoxy resins used in CFRP, and was corroborated by similarities to the carbon cluster ion distribution observed in rBC during CFRP pipe-cutting operations. Further, the particle size distribution suggested the existence of plastic aerosol particles smaller than 100 nm. This study established the method to quantitatively distinguish nano-plastic aerosol particles from other aerosol particles in high temporal resolution and these techniques are useful for accurately assessing exposure to nano-plastic aerosol particles in working environments.

Rational design for cooperative recognition of specific nucleobases using ß-cyclodextrin-modified DNAs and fluorescent ligands on DNA and RNA scaffolds.

We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a ß-cyclodextrin (ß-CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase-specific heterocycle and an environment-sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby ß-CyD. Three reporter ligands, MNDS (naphthyridine-dansyl linked ligand), MNDB (naphthyridine-DBD), and DPDB (pyridine-DBD), were used for DNA and RNA probing with 3'-end or 5'-end modified ß-CyD-ODN conjugates. For the DNA target, the ß-CyD tethered to the 3'-end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site (MNDS and DPDB). Meanwhile the ß-CyD on the 5'-end of the ODN interacted with the fluorophore in the minor groove (MNDB and DPDB). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules.

Synthesis of mesoporous metal oxide by the thermal decomposition of oxalate precursor.

A synthesis method was newly developed to prepare mesoporous transition metal oxides by thermal decomposition of transition metal oxalates, and the method was advantageous in its versatility, low cost, and environmental friendliness. Various mesoporous transition metal oxides were successfully synthesized by the newly developed method, such as magnetic γ-Fe2O3, CoFe2O4, and NiFe2O4, MnxOy, Co3O4, and NiO. Morphology, structure, and magnetic property of the synthesized mesoporous transition metal oxides were characterized by XRD, TG-DTA, SEM, TEM, quantum design SQUID, and N2 sorption techniques. From the dependency of the heating rate, calcination time, and calcination temperature on the metal oxide structures, it was revealed that the calcination temperature was the major factor to determine the final mesoporous structure of the metal oxides. The mesoporous structures were well constructed by their corresponding metal oxide nanoparticles resulting from oxalate thermal decomposition.

Base pairing at the abasic site in DNA duplexes and its application in adenosine aptasensors.

The binding of nucleosides to abasic site (AP site)-containing DNA duplexes (AP-DNAs) carrying complementary nucleosides opposite the AP site was investigated by thermal denaturation and isothermal titration calorimetric (ITC) experiments. Purine nucleosides show high affinities (K(d) =14.1 µM for adenosine and 41.8 µM for guanosine) for binding to the AP-DNAs, and the interactions are driven primarily by the enthalpy change, similarly to the case of DNA intercalators. In contrast, pyrimidine nucleosides do not show noticeable binding to the AP-DNAs, thus suggesting that stacking interaction at the AP site plays a key role in the binding of purine nucleosides to the AP-DNAs, as revealed by ITC measurements. Next, to apply an AP-DNA as an aptasensor for adenosine, a competitive assay between adenosine and AP-site-binding fluorescent ligand was performed. The assay employs a fluorescent ligand, riboflavin, that binds to the AP site in a DNA duplex, thereby causing fluorescence quenching. By adding adenosine to the riboflavin/AP-DNA complex, the binding of adenosine to the AP site causes release of riboflavin from the AP site, thereby resulting in restoration of riboflavin fluorescence. AP-DNAs can serve as a new class of aptasensors-a limit of detection of 0.7 µM was obtained for adenosine. In contrast to conventional aptasensors for adenosine, the present method shows high selectivity for adenosine over the other nucleotides (AMP, ADP and ATP). The method does not require covalent labelling of fluorophores, and thus it is cost-effective; finally, the method was successfully demonstrated to be applicable for the detection of adenosine in horse serum.

Enhanced fluorescence in a nanoporous waveguide and its quantitative analysis.

Fluorescence behavior was examined for fluorophore-labeled protein (BSA-AF) adsorbed on the nanopore surface of a nanoporous waveguiding film. The waveguiding film has a bilayer structure of a porous anodic alumina (PAA) layer on a metallic aluminum (Al) layer, and this structure allows efficient interaction of fluorophores entrapped in the nanoporous waveguiding film with a hotspot of the enhanced electromagnetic field of the waveguide modes. Fluorescence response of BSA-AF depends on the enhanced field within the waveguiding film and the enlarged adsorbed amount in the PAA layer where most of the light is confined. Enhancement of the field in the waveguiding film can be controlled by the refractive index of the PAA layer and enlargement of the pore size efficiently affects the enhancement of the fluorescence response. Compared to the film without a PAA layer, the PAA/Al film exhibits more than 140-fold larger fluorescence response due to the large adsorption capacity of the PAA nanopores and the enhanced field formed by the waveguide modes in the PAA layer with a low refractive index.

Competitive assay for theophylline based on an abasic site-containing DNA duplex aptamer and a fluorescent ligand.

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site-containing DNA duplex aptamer (AP aptamer) in combination with an abasic site-binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23-mer AP aptamer (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/5'-GTG TGC GTT GCC CTG GAC GCA GA-3'; X: the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 µM. Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.

Highly selective binding of naphthyridine with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes.

We report on highly selective binding of a naphthyridine derivative with a trifluoromethyl group to cytosine opposite an abasic site in DNA duplexes; the binding-induced fluorescence quenching is applicable to the analysis of a C-related single-base mutation in DNAs amplified by PCR.

Strong and selective binding of amiloride to an abasic site in RNA duplexes: thermodynamic characterization and microRNA detection.

Firmly tied: The binding affinity of amiloride for an abasic (AP) site-containing RNA duplex is two orders of magnitude superior to the affinity of the corresponding AP site-containing DNA duplex. The observed high binding affinity for the RNA duplex arises from a favorable enthalpy gain. The binding-induced fluorescence response of amiloride is applicable to microRNA detection.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride.

BACKGROUND: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. METHODS: The combination of experimental and theoretical methods was used. RESULTS: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. CONCLUSIONS: Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. GENERAL SIGNIFICANCE: The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena.

Acid-base equilibria inside amine-functionalized mesoporous silica.

Acid-base equilibria and effective proton concentration inside a silica mesopore modified with a trimethyl ammonium (TMAP) layer were studied by steady-state fluorescence experiments. The mesoporous silica with a dense TMAP layer (1.4 molecules/nm(2)) was prepared by a post grafting of N-trimethoxysilylpropyl-N,N,N-trimethylammonium at surfactant-templated mesoporous silica (diameter of silica framework =3.1 nm). The resulting TMAP-modified mesoporous silica strongly adsorbed of anionic fluorescence indicator dyes (8-hydroxypyrene-1,3,6-trisulfonate (pyranine), 8-aminopyrene-1,3,6-trisulfonate (APTS), 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid disulfuric acid (TPPS), 2-naphthol-3,6-disulfonate (2NT)) and fluorescence excitation spectra of these dyes within TMAP-modified mesoporous silica were measured by varying the solution pH. The fluorescence experiments revealed that the acid-base equilibrium reactions of all pH indicator dyes within the TMAP-modified silica mesopore were quite different from those in bulk water. From the analysis of the acid-base equilibrium of pyranine, the following relationships between solution pH (pH(bulk)) and the effective proton concentration inside the pore (pH(pore)) were obtained: (1) shift of pH(pore) was 1.8 (ΔpH(pore)=1.8) for the pH(bulk) change from 2.1 to 9.1 (ΔpH(bulk)=7.0); (2) pH(pore) was not simply proportional to pH(bulk); (3) the inside of the TMAP-modified silica mesopore was suggested to be in a weak acidic or neutral condition when pH(bulk) was changed from 2.0 to 9.1. Since these relationships between pH(bulk) and pH(pore) could explain the acid-base equilibria of other pH indicator dyes (APTS, TPPS, 2NT), these relationships were inferred to describe the effective proton concentration inside the TMAP-modified silica mesopore.

Label-free molecular beacon system based on DNAs containing abasic sites and fluorescent ligands that bind abasic sites.

A new class of label-free molecular beacon (MB) system based on DNA strands that contain abasic (AP) sites (AP-DNA) and adopt stem-loop structures, in combination with fluorescent ligands that bind these AP sites, has been developed. Unlike a conventional MB, which requires covalent labeling of the MB with a fluorophore and a quencher, the developed system (APMB) does not require covalent attachment of signal transduction units. Detailed sensing functions of a series of APMB systems were examined with the aid of the fluorescent ligand named ATMND to provide insight into the design strategy for APMB systems. The effects of the stem length and the position of the AP site in the stem moiety on the fluorescence response of the APMB system were examined. Genotyping of a G/C SNP of PCR amplification products was successfully demonstrated with the APMB system and blue-fluorescent ATMND as a ligand. The APMB system was further extended to a system that utilized green-fluorescent lumiflavin.

DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms.

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.

Influence of substituent modifications on the binding of 2-amino-1,8-naphthyridines to cytosine opposite an AP site in DNA duplexes: thermodynamic characterization.

Here, we report on a significant effect of substitutions on the binding affinity of a series of 2-amino-1,8-naphthyridines, i.e., 2-amino-1,8-naphthyridine (AND), 2-amino-7-methyl-1,8-naphthyridine (AMND), 2-amino-5,7-dimethyl-1,8-naphthyridine (ADMND) and 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), all of which can bind to cytosine opposite an AP site in DNA duplexes. Fluorescence titration experiments show that the binding affinity for cytosine is effectively enhanced by the introduction of methyl groups to the naphthyridine ring, and the 1:1 binding constant (10(6) M(-1)) follows in the order of AND (0.30) < AMND (2.7) < ADMND (6.1) < ATMND (19) in solutions containing 110 mM Na(+) (pH 7.0, at 20 degrees C). The thermodynamic parameters obtained by isothermal titration calorimetry experiments indicate that the introduction of methyl groups effectively reduces the loss of binding entropy, which is indeed responsible for the increase in the binding affinity. The heat capacity change (DeltaC(p)), as determined from temperature dependence of the binding enthalpy, is found to be significantly different between AND (-161 cal/mol K) and ATMND (-217 cal/mol K). The hydrophobic contribution appears to be a key force to explain the observed effect of substitutions on the binding affinity when the observed binding free energy (DeltaG(obs)) is dissected into its component terms.

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity.

BACKGROUND: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS: The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE: Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.

Properties of a metal clad waveguide sensor based on a nanoporous-metal-oxide/metal multilayer film.

A metal-clad waveguide (MCWG) sensor comprised of a nanoporous waveguiding layer on a metal cladding layer is advantageous in sensing of biomolecules because of a high surface area of nanopores and a sharp dip in the reflection spectrum due to characteristics of the MCWG mode. Here, a porous anodic alumina (PAA)/aluminum (Al) film was fabricated on a glass substrate as a MCWG sensor with the Kretschmann geometry, and the sensor response was examined for both colorless bovine serum albumin (BSA) and colored metal complexes by measurements of reflection spectra and Fresnel calculations. The BSA adsorption on the PAA layer induced a parallel redshift of the waveguide coupling dip in the reflection spectrum. The experimental results were well simulated by the five-phase Fresnel calculations which indicated that the redshift of the dip was linearly dependent on the adsorbed amount of BSA. When the response of a MCWG sensor with a PAA layer was compared with that of a MCWG sensor with a nonporous alumina layer, the former showed larger redshift than the latter, due to a large adsorbed amount of BSA in the PAA layer with high surface area. For the adsorption of colored Ru[Bphen(3)](2+) and Fe[Phen(3)](2+), the effect of both the real and imaginary parts of the complex refractive index on the sensor response was examined. As a result, a redshift of the waveguide coupling dip was observed for both metal complexes irrespective of the wavelength region examined; this could be ascribed to the changes in the real part of the refractive index due to the adsorption of metal complexes on the PAA layer. Meanwhile, an increase in the reflectivity was observed when the coupling wavelength was close to that of the absorption bands of the metal complexes; this could be ascribed to the changes in the imaginary part of the refractive index of the PAA layer. Using the sensor response caused by the changes in the imaginary part, absorption spectral profiles of metal complexes could be reproduced.