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We report successful and selective dual protein assembly on patterned bifunctional βCD-Ni(ii)NTA surfaces, using red fluorescent protein variants with hexahistidine-tags and teal fluorescent protein variants conjugated with a peptide containing three adamantyl groups. We show that dual protein patterns can only be assembled, when opposing supramolecular interactions have been optimized and nonspecific interactions have been sufficiently suppressed.

Solar-powered electrochemical production of hydrogen through water electrolysis is an active and important research endeavor. However, technologies and roadmaps for implementation of this process do not exist. In this perspective paper, we describe potential pathways for solar-hydrogen technologies into the marketplace in the form of photoelectrochemical or photovoltaic-driven electrolysis devices and systems. We detail technical approaches for device and system architectures, economic drivers, societal perceptions, political impacts, technological challenges, and research opportunities. Implementation scenarios are broken down into short-term and long-term markets, and a specific technology roadmap is defined. In the short term, the only plausible economical option will be photovoltaic-driven electrolysis systems for niche applications. In the long term, electrochemical solar-hydrogen technologies could be deployed more broadly in energy markets but will require advances in the technology, significant cost reductions, and/or policy changes. Ultimately, a transition to a society that significantly relies on solar-hydrogen technologies will benefit from continued creativity and influence from the scientific community.

Solar-powered electrochemical production of hydrogen through water electrolysis is an active and important research endeavor. However, technologies and roadmaps for implementation of this process do not exist. In this perspective paper, we describe potential pathways for solar-hydrogen technologies into the marketplace in the form of photoelectrochemical or photovoltaic-driven electrolysis devices and systems. We detail technical approaches for device and system architectures, economic drivers, societal perceptions, political impacts, technological challenges, and research opportunities. Implementation scenarios are broken down into short-term and long-term markets, and a specific technology roadmap is defined. In the short term, the only plausible economical option will be photovoltaic-driven electrolysis systems for niche applications. In the long term, electrochemical solar-hydrogen technologies could be deployed more broadly in energy markets but will require advances in the technology, significant cost reductions, and/or policy changes. Ultimately, a transition to a society that significantly relies on solar-hydrogen technologies will benefit from continued creativity and influence from the scientific community.

The modulation of the hierarchical nucleated self-assembly of tri-β3-peptides has been studied. β3-Tyrosine provided a handle to control the assembly process through host-guest interactions with CB[7] and CB[8]. By varying the cavity size from CB[7] to CB[8] distinct phases of assembling tri-β3-peptides were arrested. Given the limited size of the CB[7] cavity, only one aromatic β3-tyrosine can be simultaneously hosted and hence CB[7] was primarily acting as an inhibitor of self-assembly. In strong contrast, the larger CB[8] can form a ternary complex with two aromatic amino acids and hence CB[8] was acting primarily as cross-linker of multiple fibers and hence promoting the formation of larger aggregates. General insights on modulating supramolecular assembly can lead to new ways to introduce functionality in supramolecular polymers.

Contains fulltext : 128336.pdf (publisher's version ) (Open Access)

Contains fulltext : 128336.pdf (publisher's version ) (Open Access)

Contains fulltext : 128336.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit[7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Item does not contain fulltext, We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

Item does not contain fulltext, We report the fabrication of a patterned protein array using three orthogonal methods of immobilization that are detected exploiting a fluorogenic surface. Upon reaction of thiols, the fluorogenic tether reports the bond formation by an instantaneous rise in (blue) fluorescence intensity providing a means to visualize the immobilization even of nonfluorescent biomolecules. First, the covalent, oriented immobilization of a visible fluorescent protein (TFP) modified to display a single cysteine residue was detected. Colocalization of the fluorescence of the immobilized TFP and the fluorogenic group provided a direct tool to distinguish covalent bond formation from physisorption of proteins. Subsequent orthogonal immobilization of thiol-functionalized biomolecules could be conveniently detected by fluorescence microscopy using the fluorogenic surface. A thiol-modified nitrilotriacetate ligand was immobilized for binding of hexahistidine-tagged red-fluorescing TagRFP, while an appropriately modified biotin was immobilized for binding of Cy5-labeled streptavidin.

The supramolecular assembly of proteins on surfaces has been investigated via the site-selective incorporation of a supramolecular moiety on proteins. To this end, fluorescent proteins have been site-selectively labeled with ferrocenes, as supramolecular guest moieties, via SNAP-tag technology. The assembly of guest-functionalized SNAP-fusion proteins on cyclodextrin- and cucurbit[7]uril-coated surfaces yielded stable monolayers. The binding of all ferrocene fusion proteins is specific as determined by surface plasmon resonance. Micropatterns of the fusion proteins, on patterned cyclodextrin and cucurbituril surfaces, have been visualized using fluorescence microscopy. The SNAP-fusion proteins were also immobilized on cyclodextrin vesicles. The supramolecular SNAP-tag labeling of proteins, thus, allows for the assembly of modified proteins via supramolecular host-guest interaction on different surfaces in a controlled manner. These findings extend the toolbox of fabricating supramolecular protein patterns on surfaces taking advantage of the high labeling efficiency of the SNAP-tag with versatile supramolecular moieties.

Item does not contain fulltext, PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

1 december 2011, Item does not contain fulltext, Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

Item does not contain fulltext, PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

01 december 2011, Item does not contain fulltext, Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

01 december 2011, Item does not contain fulltext, Single-molecule force spectroscopy studies performed by Atomic Force Microscopes (AFMs) strongly rely on accurately determined cantilever spring constants. Hence, to calibrate cantilevers, a reliable calibration protocol is essential. Although the thermal noise method and the direct Sader method are frequently used for cantilever calibration, there is no consensus on the optimal calibration of soft and V-shaped cantilevers, especially those used in force spectroscopy. Therefore, in this study we aimed at establishing a commonly accepted approach to accurately calibrate compliant and V-shaped cantilevers. In a round robin experiment involving eight different laboratories we compared the thermal noise and the Sader method on ten commercial and custom-built AFMs. We found that spring constants of both rectangular and V-shaped cantilevers can accurately be determined with both methods, although the Sader method proved to be superior. Furthermore, we observed that simultaneous application of both methods on an AFM proved an accurate consistency check of the instrument and thus provides optimal and highly reproducible calibration. To illustrate the importance of optimal calibration, we show that for biological force spectroscopy studies, an erroneously calibrated cantilever can significantly affect the derived (bio)physical parameters. Taken together, our findings demonstrated that with the pre-established protocol described reliable spring constants can be obtained for different types of cantilevers.

Contains fulltext : 33788.pdf (publisher's version ) (Open Access)

Contains fulltext : 33788.pdf (publisher's version ) (Open Access)

Applied Sciences

Applied Sciences

A new deprotection procedure in the synthesis of phosphate-methylated oligodeoxynucleotides involves treatment of fully protected DNA fragments with methanolic K2CO3. This reagent, for example, deblocks 9-fluorenylmethoxycarbonyl-protected bases and cyanomethyl-protected phosphotriesters and effectively removes the DNA oligomer from the controlled-pore glass support. Thus, DNA fragments which are phosphate-methylated at defined positions may be synthesized both in soln. and on a solid support.

A new deprotection procedure in the synthesis of phosphate-methylated oligodeoxynucleotides involves treatment of fully protected DNA fragments with methanolic K2CO3. This reagent, for example, deblocks 9-fluorenylmethoxycarbonyl-protected bases and cyanomethyl-protected phosphotriesters and effectively removes the DNA oligomer from the controlled-pore glass support. Thus, DNA fragments which are phosphate-methylated at defined positions may be synthesized both in soln. and on a solid support.