This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. IM release LLNL-JRNL-750634.

CAUTION: Please consult with the safety data sheets (SDS) of all materials, and wear proper protective equipment (PPE) when handling materials before casting and curing. Resorcinol and polyethylene imine are known to be toxic. Formaldehyde is both toxic and carcinogenic20. Preparation of ceramic suspensions should be done in chemical fume hoods or other properly ventilated work environments.
1. Negative Additive Manufacturing
<ol>
	<li>Preparation of a 120 mL two-part suspension 
	NOTE: A two-part suspension will be prepared to help prolong the shelf life of the suspensions before casting. One suspension (R-mix) will contain the resorcinol component, and the other (F-mix) will contain the formaldehyde component. Both suspensions will be mixed together to form a final suspension that will initiate the gelation process.
	<ol>
		<li>To create the R-mix, begin by dissolving 0.88 g of polyethylene imine (PEI) in 25.00 g of water using a planetary mixer.</li>
		<li>To create a separate F-mix, dissolve 0.88 g of polyethylene imine (PEI) in 16.83 g of water using a planetary mixer. 
		NOTE: Using a planetary mixer at 2000 rpm for at least several minutes will provide sufficient shearing forces to help dissolve the viscous PEI, resorcinol, and formaldehyde, and to suspend the boron carbide particles. PEI serves as the dispersing agent for the B4C particles</li>
		<li>Dissolve 12.60 g of resorcinol powder into the R-mix. The solution should turn from a cloudy-white to a clear transparent solution after complete dissolution of the powder from mixing.</li>
		<li>Add 17.03 g of formaldehyde solution to the F-mix and ensure complete mixing.</li>
		<li>Incrementally add 5.25 g (12 increments until reaching 63.00 g) of boron carbide powder (1500F) into both the R-mix and F-mix separately.</li>
		<li>Add 6.50 g of acetic acid to the R-mix and F-mix and ensure complete mixing in each. 
		NOTE: At this point, the two-part suspensions will have 42 vol% of B4C and are ready to be combined for casting or stored for future use (if adequately sealed). Beware that if the suspensions sit for ~1 h or more, particle settling will occur. Ensure that the particles are resuspended by applying thorough agitation before using the suspensions. Also, three different commercial batches of boron carbides, 1250F, 1500F, and 3000F (named according to their approximate sieved mesh sizes), were originally tested. Each batch has a different particle size distribution, and the 1500F B4C batch was found to achieve the highest sintering density, as reported in Lu et al.8. Acetic acid can also be added before the B4C solids loading step as well, but adding at the end offers better ease of handling by limiting acetic acid odors.</li>
	</ol>
	</li>
	<li>Preparation of the 3D printed molds for casting
	<ol>
		<li>Prepare the mold design in a computer-aided design (CAD) software program.</li>
		<li>Print the molds using a Fused Deposition Modeling (FDM) 3D printer with acrylonitrile butadiene styrene (ABS) filaments. 
		NOTE: Acetone vapors can be used to smooth out the mold texture if desired21. The suggested nozzle and bed temperatures are 240 &#176;C and 110 &#176;C, respectively. Parameters such as layer thickness (0.2 mm), extrusion speed, and cooling rate are chosen to optimize the quality of the part with minimum deformations. This requires some trial and error with each unique printer system. A wall thickness of at least 1 mm is advised. The minimum feature size is 0.5 mm; however, it is suggested not to go below 1 mm. Molds from Lu et al.8 are available for download online in supporting material.</li>
	</ol>
	</li>
	<li>Combination of the two-part suspension to prepare for casting
	<ol>
		<li>Before combining, thoroughly agitate (by using a vortex or planetary mixer) the R-mix with the F-mix suspensions individually to ensure the B4C particles are well-suspended.</li>
		<li>Combine the R-mix and F-mix to obtain the final suspension. 
		NOTE: The pH of the combined suspension should be 2.8, which will provide about 30 minutes of working time to de-air and cast the final suspension before gelation starts occurring. The onset of gelation can be observed from the sharp increase in viscosity of the suspension.</li>
		<li>Before casting, mix and apply vacuum (20-200 torr or 2.7-27 kPa) to the final suspension mixture for about 10 minutes to remove air bubbles without boiling the water. This can be accomplished by using a stirring plate at 200-300 rpm with a vacuum jar.</li>
	</ol>
	</li>
	<li>Gelcasting
	<ol>
		<li>Immediately pour the de-aired suspension into the 3D-printed molds.</li>
		<li>Place the molds inside a sealed glass container to prevent moisture loss during the curing process.</li>
		<li>Place the sealed container with the molds into a 60-80 &#176;C oven to initiate the curing process.</li>
		<li>Allow the casts to cure for at least 8 hours for parts that are several centimeters in length scale or possibly longer for larger molds.</li>
	</ol>
	</li>
	<li>Dissolution of the molds to obtain green bodies
	<ol>
		<li>Remove the sealed container with the molds from the oven and allow it to cool to room temperature.</li>
		<li>Add enough acetone into the container until the mold is fully submersed. The amount will vary depending on the size and volume of the mold used (generally ~100 mL of acetone for a mold that is 50 cm3 in dimension). 
		NOTE: This process may take up to 2-4 days depending on the volume of plastic that needs to be dissolved away. Minimal agitation of the acetone bath or heating it slightly to 40 &#176;C may help speed up the process. Execute caution when heating acetone bath, as it is a flammable chemical and may become explosive when combined with air in certain composition ranges.</li>
		<li>Extract the free green body from the acetone bath after the ABS plastic is dissolved away. 
		NOTE: After the RF is cured, the mold can be dissolved away to obtain a solid green body shaped as a negative copy of the inner mold geometry. This green body should be strong enough to survive gentle and careful handling in the subsequent post-processing steps without breaking.</li>
		<li>Place the green bodies in an oven at 80 &#176;C to ensure complete drying and removal of all moisture. 
		NOTE: Drying time varies depending on the volume of the green body. Leaving the part to dry overnight (&#62; 8 hours) is sufficient for green body sizes less than 1000 cm3. There is no harm in over-drying.</li>
	</ol>
	</li>
</ol>
2. Carbonization
<ol>
	<li>After drying, place each green body in a 2-inch quartz tube lined with graphite foil and put them into a furnace with flowing gas [250 standard cubic centimeters of air (SCCM) consisting of 4 wt% H2(g) and 96 wt% Ar(g) to create a reducing atmosphere during the pyrolysis treatment].</li>
	<li>Heat the green bodies inside the furnace at 5 &#176;C/min until 1050 &#176;C and hold for 3 hours. 
	NOTE: The gel-cast green bodies will have 15 wt% of RF relative to the B4C and will provide about 7.5 wt% in carbon after the pyrolysis process. This process removes much of the resorcinol-formaldehyde residue and will severely contaminate the furnace if no trap is used.</li>
	<li>Ensure that the green bodies come out uniformly darker in color, indicating the presence of carbon from the pyrolysis treatment.</li>
</ol>
3. Sintering
NOTE: After sintering, the surface roughness of the samples will improve slightly compared to the surface roughness of the molds used. This is a consequence of the 57-58 vol% shrinkage of the samples from sintering.
<ol>
	<li>Place the carbonized parts in a graphite furnace with vacuum backfilled flowing helium gas (420 SCCM) for sintering. Apply 280 SCCM to the front and pyrometer windows and 140 SCCM directly into the sample chamber with an inlet pressure of ~170 kPa.</li>
	<li>Heat up the furnace to 2290 &#176;C (20 K/min to 2000 &#176;C then 3 K/min to 2290 &#176;C) and hold for 1 hour to achieve optimal densification of the parts. 
	NOTE: Archimedes density is a common and quick technique to measure the density of the sintered boron carbide parts. Archimedes density kits can be added onto analytical balance scales to measure the density of samples or manually determined22. Boron carbide with 7.5 wt% in carbon will have a theoretical maximum density (TMD) of 2.49 g/cm3. Parts sintered at 2290 &#176;C from this methodology will result in 2.43 &#177; 0.01 g/cm3 which is 97.6 &#177; 0.4% TMD.</li>
</ol>

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The authors have nothing to disclose.

The methodology of negative additive manufacturing described in the protocol allows complex shaped boron carbide parts to be produced at nearly full density after sintering at an optimal temperature of 2290 &#176;C. The first several steps related to preparation and casting are the most critical for generating a high-quality cast with minimal defects. If the viscosity of the suspension is too high, poor mixing will occur. The porosity of the sintered part is also affected since increased viscosity hinders air bubble remo...

Boron carbide (B4C), with a Vickers hardness of about 38 GPa, is known as the third hardest commercially available material, behind diamond (~115 GPa) and cubic boron nitride (~48 GPa). This particular property, along with a low density (2.52 g/cm3), makes it attractive for defense applications such as armors1. B4C also has a high melting point, superior wear resistance, and high neutron absorption cross section2,3,4. However, utilization of these favorable mechanical properties typically requires B4C to be sintered to a high density. Hot pressing is a conventional method for sintering B4C to full densification. This technique is often limited to simple geometries with limited curvature and fairly uniform thickness. Expensive and labor-intensive machining with polycrystalline diamond tooling or laser cutting is required to introduce finer or more complex features.
Alternatively, colloidal forming techniques with pressure-less sintering can produce near-full density parts that require minimal to no machining. Due to a lack of external pressure during consolidation, sintering aids are normally added to the ceramic medium to increase the effectiveness of pressureless sintering. Carbon is commonly used as a sintering aid for B4C5,6,7.Various carbon sources, such as nanoparticle powders or carbonized organics from pyrolysis, can be used. Homogeneous distribution of the carbon sintering aid along grain boundaries is an important factor for obtaining uniform sintering of B4C. Therefore, carbon concentration and B4C particle size are also important and interrelated factors for sintering parts to high density8.
One of the most promising colloidal forming techniques for obtaining complex shaped ceramic parts is gelcasting. This technique involves casting a ceramic suspension with an organic monomer into a mold which polymerizes in situ to act as a gel9,10,11. The gel serves as a binder to form a green body in the shape of the mold that is strong enough to be handled without breakage in subsequent processing steps. Previously impossible 3D mold geometries can now be produced through low-cost polymer-based additive manufacturing (AM) techniques such as stereolithography (SLA) and fused deposition modeling (FDM)12. The recent availability of 3D printers has opened new possibilities for designing ceramics with highly complex geometries.
Negative additive manufacturing is a technique that combines gelcasting with sacrificial 3D-printed molds. The complexity of the ceramic part is directly related to the complexity of the mold design. Mold designs can now be incredibly sophisticated with the advent of high resolution plastic 3D printers. For example, 3D scanning tools can be used to capture an individual&#39;s contours and be incorporated into molds. By using negative AM, lightweight ceramic armors tailored to the individual&#39;s body size and shape can be created. Such design customizations can provide lighter weight armors with enhanced mobility for users.
Other common ceramic AM techniques such as direct ink write (DIW), selective laser sintering (SLS), and binder jetting (BJ) are also effective in producing complex shaped ceramic parts. However, most of these techniques are only useful for producing fine porous structures and are not efficient when scaling up to large parts, such as armor applications13,14,15,16,17. Moreover, most of these techniques are not feasible for high volume production due to high expenses. Therefore, negative AM is a preferred and relatively inexpensive route for industrial-level production of large-scale parts.
The B4C suspensions used for gelcasting must be low in viscosity and contain a gelling agent and sintering aid. Resorcinol and formaldehyde are chosen for their ability to undergo polycondensation reactions to form a resorcinol-formaldehyde (RF) network, which helps to bind the B4C particles together. Traditional hydrogels used for gelcasting are limited to molds with hollow cores due to the high inward shrinkage experienced during the drying process18. Since RF is commonly used as an aerogel, there is little to no shrinkage, which permits the use of more intricately shaped molds. Another advantage of using RF is that the gelation rate can be controlled by altering the pH of the suspension&#160;(Figure 3). Additionally, suspensions containing either resorcinol or formaldehyde can be prepared in advanced and stored separately until they are ready for casting. Most importantly, the RF gel can be pyrolyzed to leave behind 50 wt% carbon19. This highly homogenous distribution of carbon can aid the densification of B4C to near-full densities during sintering. 15 wt% of RF relative to boron carbide is used in the formulation of the suspension to provide 7.5 wt% of carbon after pyrolysis of the cast parts.
The overall goal of this work is to combine traditional gelcasting techniques with inexpensive 3D printing capabilities and a unique gelling agent to obtain near-full density boron carbide parts with highly complex geometries. In addition to ceramics, negative AM can be applied to other material fields to create entirely new geometries of multi-material systems. The methodology described here expands on the work presented in Lu et al.8&#160;and aims to provide a more detailed protocol for reproducing those results.

<table border="0" cellpadding="0" cellspacing="0" style="width:1045px;" width="1044">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:215px;">Boron carbide powder 1250F</td>
			<td style="width:253px;">Tetrabor Ceramics</td>
			<td style="width:215px;">Lot 211M419</td>
			<td style="width:363px;">&#62;96% purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Boron carbide powder 1500F</td>
			<td style="width:253px;">Tetrabor Ceramics</td>
			<td style="width:215px;">Lot 209M102/9</td>
			<td>&#62;96% purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Boron carbide powder 3000F</td>
			<td style="width:253px;">Tetrabor Ceramics</td>
			<td>Lot 111m53/9&#160;</td>
			<td>&#62;96% purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polyethylene Imine (PEI)</td>
			<td style="width:253px;">Sigma Aldrich</td>
			<td style="width:215px;">MKBP3417V</td>
			<td>Averaged MW ~25,000 by L.S.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Resorcinol</td>
			<td style="width:253px;">Sigma Aldrich</td>
			<td style="width:215px;">MKBG6751V</td>
			<td>BioXtra, &#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Formaldehyde</td>
			<td style="width:253px;">Fisher Scientific</td>
			<td style="width:215px;">F79-1</td>
			<td>37% by weight; Stabilized with 10-15% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acetic Acid</td>
			<td style="width:253px;">Sigma Aldrich</td>
			<td style="width:215px;">SKU 695092</td>
			<td>Glacial &#8805;99.7%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acetone</td>
			<td style="width:253px;">Sigma Aldrich</td>
			<td>SKU 179124</td>
			<td>ACS Reagent Grade &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Water</td>
			<td style="width:253px;">LLNL In-house (Milli-Q)</td>
			<td style="width:215px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Planetary Mixer</td>
			<td style="width:253px;">Thinky</td>
			<td style="width:215px;">AR-250</td>
			<td>Fits 150mL and 300mL Thinky containers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Acrylonitrile butadiene styrene (ABS) plastic filament</td>
			<td style="width:253px;">eSUN</td>
			<td style="width:215px;"></td>
			<td>Natural color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Taz 6 (3D printer)</td>
			<td style="width:253px;">Lulzbot</td>
			<td style="width:215px;"></td>
			<td>FDM 3D printer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">4%H2/96%Ar gas</td>
			<td style="width:253px;">Air Gas</td>
			<td style="width:215px;">UHP</td>
			<td>4% Hydrogen, balanced Argon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Helium gas</td>
			<td style="width:253px;">Air Gas</td>
			<td style="width:215px;">UHP</td>
			<td>Helium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Heating oven</td>
			<td style="width:253px;">Neytech</td>
			<td style="width:215px;">Vulcan 9493308</td>
			<td>Oven for 80 &#176;C curing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Quartz tube furnace</td>
			<td style="width:253px;">Applied Test Systems, Inc.&#160;</td>
			<td style="width:215px;">LEA 05-000075</td>
			<td>Furnace for 1050 &#176;C carbonization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Graphite furnace</td>
			<td style="width:253px;">Thermal Technology LLC</td>
			<td style="width:215px;"></td>
			<td>Sintering furnace</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Scanning Electron Microscope (SEM)</td>
			<td style="width:253px;">Jeol</td>
			<td style="width:215px;">JSM-7401F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">pH meter</td>
			<td style="width:253px;">Thermo Scientific</td>
			<td style="width:215px;">Orion 4 Star</td>
			<td>calibrated with buffer standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Rheometer</td>
			<td style="width:253px;">TA Instrument</td>
			<td style="width:215px;">AR2000ex</td>
			<td>For measurement of viscosity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">X-ray Diffractometer (XRD)</td>
			<td style="width:253px;">Bruker</td>
			<td style="width:215px;">AX D8 Advanced</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Analytical balance</td>
			<td style="width:253px;">Mettler Toledo</td>
			<td style="width:215px;">XS104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bruker EVA&#160;</td>
			<td style="width:253px;"></td>
			<td style="width:215px;"></td>
			<td>XRD Analysis Software</td>
		</tr>
	</tbody>
</table>

negative additive manufacturing of complex shaped boron carbides

Boron carbide (B4C) is one of the hardest materials in existence. However, this attractive property also limits its machineability into complex shapes for high wear, high hardness, and lightweight material applications such as armors. To overcome this challenge, negative additive manufacturing (AM) is employed to produce complex geometries of boron carbides at various length scales. Negative AM first involves gelcasting a suspension into a 3D-printed plastic mold. The mold is then dissolved away, leaving behind a green body as a negative copy. Resorcinol-formaldehyde (RF) is used as a novel gelling agent because unlike traditional hydrogels, there is little to no shrinkage, which allows for extremely complex molds to be used. Furthermore, this gelling agent can be pyrolyzed to leave behind ~50 wt% carbon, which is a highly effective sintering aid for B4C. Due to this highly homogenous distribution of in situ carbon within the B4C matrix, less than 2% porosity can be achieved after sintering. This protocol highlights in detail the methodology for creating near fully dense boron carbide parts with highly complex geometries.

Following the outlined procedure (Figure 1), complex shaped boron carbide parts with carbon (B4C/C) can be sintered up to 97.6 &#177; 0.4% of theoretical max density with a Vicker&#39;s hardness of 23.0 &#177; 1.8 GPa8. Several possible examples of sintered B4C/C parts are demonstrated (Figure 2). These examples show the fine textural features that can be copied by the gelcasting tech...

Watch this Scientific Journal Video about Negative Additive Manufacturing of Complex Shaped Boron Carbides at JoVE.com

Negative Additive Manufacturing of Complex Shaped Boron Carbides

A method called negative additive manufacturing is used to produce near fully dense complex shaped boron carbide parts of various length scales. This technique is possible via the formulation of a novel suspension involving resorcinol-formaldehyde as a unique gelling agent that leaves behind a homogenous carbon sintering aid after pyrolysis.

Boron carbide (B4C) is one of the hardest materials in existence. However, this attractive property also limits its machineability into complex shapes for ...

negative-additive-manufacturing-of-complex-shaped-boron-carbides

Research

JoVE Journal

Chemistry

8.5K Views.  Lawrence Livermore National Laboratory. A method called negative additive manufacturing is used to produce near fully dense complex shaped boron carbide parts of various length scales. This technique is possible via the formulation of a novel suspension involving resorcinol-formaldehyde as a unique gelling agent that leaves behind a homogenous carbon sintering aid after pyrolysis.

Video: Negative Additive Manufacturing of Complex Shaped Boron Carbides

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Atomically Defined Templates for Epitaxial Growth of Complex Oxide Thin Films

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain such surfaces are described. The first approach is the preparation of single terminated perovskite SrTiO3 (001) and DyScO3 (110) substrates. Wet etching was used to selectively remove one of the two possible surface terminations, while an annealing step was used to increase the smoothness of the surface. The resulting single terminated surfaces allow for the heteroepitaxial growth of perovskite oxide thin films with high crystalline quality and well-defined interfaces between substrate and film. In the second approach, seed layers for epitaxial film growth on arbitrary substrates were created by Langmuir-Blodgett (LB) deposition of nanosheets. As model system Ca2Nb3O10- nanosheets were used, prepared by delamination of their layered parent compound HCa2Nb3O10. A key advantage of creating seed layers with nanosheets is that relatively expensive and size-limited single crystalline substrates can be replaced by virtually any substrate material.

Various procedures are outlined to prepare atomically defined templates for epitaxial growth of complex oxide thin films. Chemical treatments of single crystalline SrTiO3 (001) and DyScO3 (110) substrates were performed to obtain atomically smooth, single terminated surfaces. Ca2 Nb3 O10- nanosheets were used to create atomically defined templates on arbitrary substrates.

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain ...

Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles.

Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...