To date, aggregation of DNA-functionalized gold nanoparticles by hybridization of target DNA in a cross-linking configuration has been intensively studied. Here, we report that aggregation in a non-cross-linking configuration is also possible and is even better from the viewpoint of genetic analysis because of its speed and sensitivity. In this system, 15 nm diameter gold nanoparticles functionalized with (alkanethiol)-15mer DNA are hybridized to target 15mer DNA at room temperature. At high NaCl concentration (>/=0.5 M), hybridization with complementary target DNA induces nanoparticle aggregation based on the salting-out effect. The aggregation can be detected by a colorimetric change of the colloidal solution within 3 min. Furthermore, unusual sensitivity of this system for single-base mismatch at the terminus opposite to the anchored side has been discovered. In fact, target DNA with such a kind of mismatch does not induce the colorimetric change at all, while target DNA with single-base mismatch at the middle of it cannot be discriminated from the fully complementary target. This non-cross-linking aggregation system opens up a new possibility of rapid and reliable genetic analysis.

The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel beta-sheet structure, whereas the insulin fibrils have a parallel beta-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.

Molecule-responsive hydrogels are reputed to be smart materials because of their unique properties. We recently reported that hydrogels containing directly grafted single-stranded (ss) DNA or ssDNA-polyacrylamide conjugate in a semi-interpenetrating network (semi-IPN) manner that "only shrunk" by the addition of ssDNA samples. To date, however, no DNA-responsive hydrogels have been reported capable of "swelling" in response to specific DNAs. Smart materials capable of both shrinking and swelling in response to specific DNAs would be very useful in biochemical and biomedical applications. Here, we show a novel "shrinking or swelling" DNA-responsive mechanism. Novel hybrid hydrogels containing rationally designed ssDNA as the cross-linker were capable of shrinking or swelling in response to ssDNA samples and recognizing a single base difference in the samples. On the basis of the results presented in this paper, it is envisioned that these novel hybrid hydrogels could function and have potential in applications such as DNA-sensing devices and DNA-triggered actuators.

This paper presents a simple fluid handling technique for microchip immunoassay. Necessary solutions were sequentially injected into a microchannel by air-evacuated poly(dimethylsiloxane), and were passively regulated by capillary force at the inlet opening. For heterogeneous immunoassay, microchips are potentially useful for reduction of sample consumption and assay time. However, most of the previously reported microchips have limitations in their use because of the needs for external power sources for fluid handling. In this paper, an on-chip heterogeneous immunofluorescence assay without such an external power source is demonstrated. The microchip consisting of poly(dimethylsiloxane) (PDMS) and glass has a simple structure, and therefore is suitable for single-use applications. Necessary solutions were sequentially injected into a microchannel in an autonomous fashion with the power-free pumping technique, which exploits the high solubility and the rapid diffusion of air in PDMS. For deionized water, this method yielded flow rates of 3-5 nL s-1 with reproducibility of 4-10%. The inlet opening of the microchannel functioned as a passive valve to hold the solution when the flow was finished. Rabbit immunoglobulin G (rIgG) and human C-reactive protein (CRP) were detected using the microchannel walls as reaction sites. With the sample consumption of 1 microL and the assay time of approximately 20 min including the antibody immobilization step, the sandwich immunoassay methods for rIgG and CRP exhibited the limits of detection of 0.21 nM (0.21 fmol) and 0.42 nM (0.42 fmol), respectively.

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Cell-culturing substrates where cell adhesion can be switched on by external stimuli during cell cultivation are useful scaffolds for tissue engineering, cell-based drug screening, and fundamental cellular studies. Here, we show a new strategy for photoactivation of a substrate for cell adhesion under standard fluorescence microscopes. A glass substrate chemically modified with an alkylsiloxane having a photocleavable 2-nitrobenzyl group was coated with bovine serum albumin to prevent cell adhesion. Upon irradiation under a fluorescence microscope, the protein was replaced with fibronectin, which made the irradiated region cell-adhesive. Subsequent seeding of HEK293 or COS7 cells produced patterns corresponding to the irradiated patterns. We succeeded for the first time in positioning single cells in proximity to cultivating single cells. The present method provides a general strategy for positioning single cells of same or different types at any locations on the substrate and will be useful for studying cell-cell interactions.

An extremely simple, power-free pumping method for poly(dimethylsiloxane)(PDMS) microfluidic devices is presented. By exploiting the high gas solubility of PDMS, the energy for the pumping is pre-stored in the degassed bulk PDMS, therefore no additional structures other than channels and reservoirs are required. In a Y-shaped microchannel with cross section of 100 microm width x 25 microm height, this method has provided flow rate of 0.5-2 nL s(-1), corresponding to linear velocity of 0.2-0.8 mm s(-1), with good reproducibility. As an application of the power-free pumping, gold nanoparticle-based DNA analysis, which does not rely on the cross-linking mechanism between nanoparticles, has been implemented in a microchannel with three inlets. Target 15mer DNA has been easily and unambiguously discriminated from its single-base substituted mutant. Instead of colorimetric detection in a conventional microtube, an alternative detection technique suitable for microdevices has been discovered-observation of deposition on the PDMS surfaces. The channel layout enabled two simultaneous DNA analyses at the two interfaces between the three laminar streams.