Enzyme digestion of entrapped single-DNA molecules in nanopores

The real-time digestion of entrapped single-DNA molecules by λ-exonuclease in nanoporous alumina membranes was observed using an epifluorescence microscope. The alumina membrane provides pL (∼10⁻¹² L) containers for confining single-DNA molecules without immobilization. When one end of the DNA molecule was inserted into a nanopore, it was possible to monitor the digestion process outside, near and inside the pore, where the individual DNA molecules exhibited different characteristic digestion modes. The digestion rates calculated from the decrease in fluorescence intensity showed different values according to the location of the individual molecules. Entrapment rather than immobilization allows the DNA strand to be fully exposed to the enzyme and the reaction buffer. These results confirm that the enzymatic digestion of DNA molecules is affected by their three-dimensional (3D) environment.     

Production of Lactate in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Redox Regulation Genetically and Physiologically

Escherichia coli grows fermentatively in glucose-containing medium under anaerobic condition with formation of a mixture of organic acids (lactate, acetate, formate, and succinate) and ethanol to accommodate reducing equivalents generated during glycolysis. In this paper, we tried to improve the lactate accumulation in E. coli by redox regulation genetically and physiologically. Our results indicated that genetic regulation of the host by reducing the reductive by-product may improve the lactate production. In addition, lactate accumulation was also improved under reduced and anaerobic cultivation conditions. Engineered E. coli SDU4 was able to accumulate lactate under strictly anaerobic conditions to 100 g/L with a yield of 1.97 mol/mol glucose.     

Synthesis and X-ray diffraction analysis/crystal structure of new germatranes containing methyl substituents in three five-membered chelating rings

Three monomeric germatranes, 1-isopropoxy-3,3,7,7,10,10-hexamethyl-2,8,9-trioxa-5-aza-1-germatricyclo[3.3.3.0^1,5]undecane (1), 1-isopropoxy-3,3,7,7-tetramethyl-2,8,9-trioxa-5-aza-1-germatricyclo[3.3.3.0^1,5]undecane (2), and 1-isopropoxy-3,3-dimethyl-2,8,9-trioxa-5-aza-1-germatricyclo[3.3.3.0^1,5]undecane (3) have been synthesized by the reaction of Ge(O-i-Pr)₄ in refluxing toluene with corresponding triethanolamines, (HOCH₂CH₂)_nN(CH₂CMe₂OH)_3−n (n = 0, L1H₃; n = 1, L2H₃; n = 2, L3H₃), where the number of CMe₂ groups adjacent to a OH functionality varied from 3 (L1H₃) to 2 (L2H₃), and to 1 (L3H₃). These germatranes 1-3 have been characterized by solution ¹H and ¹³C{¹H} NMR and the solid state structure of 2 has been determined by single crystal X-ray diffraction.     

Au nanowire-on-film SERRS sensor for ultrasensitive Hg2+ detection

We report an ultrasensitive and selective single nanowire-on-film (SNOF) surface-enhanced resonance Raman scattering (SERRS) sensor for Hg(2+) detection based on structure-switching double stranded DNAs (dsDNAs). Binding of Hg(2+) induces conformational changes of the dsDNAs and let a Raman reporter get close to the SNOF structure, thereby turning on SERRS signal. The well-defined SNOF structure provides a detection limit of 100 pM with improved accuracy in Hg(2+) detection. This sensor is stable over a considerable amount of time and reusable after simple treatment. Since this SNOF sensor is composed of a single Au NW on a film, development of a multiplex sensor would be possible by employing NWs modified by multiple kinds of aptamers.     

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.     

Detection of single nucleotide polymorphisms by a gold nanowire-on-film SERS sensor coupled with S1 nuclease treatment

Single nucleotide polymorphisms (SNPs) can serve as important biomarkers for genetic diseases, for which accurate detection of SNPs is essential for early diagnosis. We have developed a novel SNP sensor by combining a Au nanowire-on-film surface-enhanced Raman scattering (SERS) platform with S1 nuclease reaction. The combined sensor system provides reproducible SERS signals only in the presence of perfectly matched target DNAs, to probe DNAs as a result of single-stranded DNA-specific degradation by S1 nuclease. Furthermore, point mutations in DNA causing Wilson disease and Avellino corneal dystrophy were successfully identified by this sensor, thereby indicating its practical ability to diagnose genetic diseases.     

Chemical and thermal stability of isotypic metal-organic frameworks: effect of metal ions

Chemical and thermal stabilities of isotypic metal-organic frameworks (MOFs) like Al-BDC (Al-benzenedicarboxylate called MIL-53-Al), Cr-BDC (MIL-53-Cr) and V-BDC (MIL-47-V), after purification to remove uncoordinated organic linkers, have been compared to understand the effect of the central metal ions on the stabilities of the porous MOF-type materials. Chemical stability to acids, bases, and water decreases in the order of Cr-BDC>Al-BDC>V-BDC, suggesting stability increases with increasing inertness of the central metal ions. However, thermal stability decreases in the order of Al-BDC>Cr-BDC> V-BDC, and this tendency may be explained by the strength of the metal-oxygen bond in common oxides like Al(2)O(3), Cr(2)O(3), and V(2)O(5). In order to evaluate precisely the stability of a MOF, it is necessary to remove uncoordinated organic linkers that are located in the pores of the MOF, because a filled MOF may be more stable than the same MOF after purification.