Name: self-assembly of hybrid lipid membranes doped with hydrophobic organic molecules at the water/air interface
Uploaded: 2020-05-01T14:00:00
Description: Watch this Scientific Journal Video about Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface at JoVE.com

This work was supported by the CREST program of the Japan Science and Technology Agency (JPMJCR14F3) and Grant in-Aids from Japan Society for the Promotion of Science (19H00846 and 18K14120). This work was partly carried out at the Laboratory for Nanoelectronics and Spintronics, Research Institute of Electrical Communication, Tohoku University.

1. Preparation of a hybrid solution
<ol>
	<li>Wash four 4 mL disposable glass vials and screw caps (with PTFE coated seals) in an ultrasonic bath for 10 min in distilled water (purified with a filtration system), followed by ethanol and chloroform, respectively. Dry the glass vials and caps in a stream of nitrogen gas.</li>
	<li>In an anaerobic glove box, prepare a CuPc stock solution (10 mg/mL) in a washed glass vial by dissolving powdered CuPc in chloroform.</li>
	<li>Filter the CuPc solution through a 0.2 &#181;m po...

<ol>
	<li>Israelachvili, J. N., Mitchell, D. J., Ninham, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+self-assembly+of+lipid+bilayers+and+vesicles.">Theory of self-assembly of lipid bilayers and vesicles.</a> Biochimica Et Biophysica Acta-Biomembranes. 470 (2), 185-201 (1977).</li><li>Venable, R. M., Zhang, Y., Hardy, B. J., Pastor, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dynamics+simulations+of+a+lipid+bilayer+and+of+hexadecane:+an+investigation+of+membrane+fluidity.">Molecular dynamics simulations of a lipid bilayer and of hexadecane: an investigation of membrane fluidity.</a> Science. 262 (5131), 223-226 (1993).</li><li>Ide, T., Ichikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+artificial+lipid-bilayer+formation.">A novel method for artificial lipid-bilayer formation.</a> Biosensors and Bioelectronics. 21 (4), 672-677 (2005).</li><li>Funakoshi, K., Suzuki, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+formation+by+contacting+monolayers+in+a+microfluidic+device+for+membrane+protein+analysis.">Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis.</a> Analytical Chemistry. 78 (24), 8169-8174 (2006).</li><li>Kongsuphol, P., Fang, K. B., Ding, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+bilayer+technologies+in+ion+channel+recordings+and+their+potential+in+drug+screening+assay.">Lipid bilayer technologies in ion channel recordings and their potential in drug screening assay.</a> Sensors and Actuators B: Chemical. 185, 530-542 (2013).</li><li>Demarche, S., Sugihara, K., Zambelli, T., Tiefenauer, L., Voros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+recording+reconstituted+ion+channels.">Techniques for recording reconstituted ion channels.</a> Analyst. 136 (6), 1077-1089 (2011).</li><li>Sakaguchi, N., Kimura, Y., Hirano-Iwata, A., Ogino, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Au-nanoparticle-embedded+lipid+bilayer+membranes+supported+on+solid+substrates.">Fabrication of Au-nanoparticle-embedded lipid bilayer membranes supported on solid substrates.</a> The Journal of Physical Chemistry B. 121 (17), 4474-4481 (2017).</li><li>Schulz, M., Olubummo, A., Binder, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+lipid+bilayer:+interaction+of+polymers+and+nanoparticles+with+membranes.">Beyond the lipid bilayer: interaction of polymers and nanoparticles with membranes.</a> Soft Matter. 8 (18), 4849-4864 (2012).</li><li>Wang, J., Wei, Y., Shi, X., Gao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+entry+of+graphene+nanosheets:+the+role+of+thickness,+oxidation+and+surface+adsorption.">Cellular entry of graphene nanosheets: the role of thickness, oxidation and surface adsorption.</a> RSC Advances. 3 (36), 15776-15782 (2013).</li><li>Vo&#776;gele, M., Ko&#776;finger, J., Hummer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dynamics+simulations+of+carbon+nanotube+porins+in+lipid+bilayers.">Molecular dynamics simulations of carbon nanotube porins in lipid bilayers.</a> Faraday Discussions. 209, 341-358 (2018).</li><li>Kanomata, K., Deguchi, T., Ma, T., Haseyama, T., Miura, M., Yamaura, D., Tadaki, D., Niwano, M., Hirano-Iwata, A., Hirose, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomodulation+of+electrical+conductivity+of+a+PCBM-doped+free-standing+lipid+bilayer+in+buffer+solution.">Photomodulation of electrical conductivity of a PCBM-doped free-standing lipid bilayer in buffer solution.</a> Journal of Electroanalytical Chemistry. 832, 55-58 (2019).</li><li>Barnoud, J., Rossi, G., Monticelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Membranes+as+Solvents+for+Carbon+Nanoparticles.">Lipid Membranes as Solvents for Carbon Nanoparticles.</a> Physical Review Letters. 112, 068102 (2014).</li><li>Dichello, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+containing+small+gold+nanoparticles+using+electrostatic+interactions.">Preparation of liposomes containing small gold nanoparticles using electrostatic interactions.</a> European Journal of Pharmaceutical Sciences. 105, 55-63 (2017).</li><li>Sullivan, P., Heutz, S., Schultes, S. M., Jones, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+codeposition+on+the+performance+of+CuPc&#8722;C60+heterojunction+photovoltaic+devices.">Influence of codeposition on the performance of CuPc&#8722;C60 heterojunction photovoltaic devices.</a> Applied Physics Letters. 84 (7), 1210-1212 (2004).</li><li>Miyata, T., Kawaguchi, S., Ishii, M., Minami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+chlorine+gas+sensors+using+Cu&#8722;phthalocyanine+thin+films.">High sensitivity chlorine gas sensors using Cu&#8722;phthalocyanine thin films.</a> Thin Solid Films. 425 (1-2), 255-259 (2003).</li><li>Barrena, E., de Oteyza, D. G., Dosch, H., Wakayama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+supramolecular+self-assembly+of+binary+organic+monolayers.">2D supramolecular self-assembly of binary organic monolayers.</a> ChemPhysChem. 8 (13), 1915-1918 (2007).</li><li>Xiao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-induced+orientation+control+of+CuPc+molecules+for+the+epitaxial+growth+of+highly+ordered+organic+crystals+on+graphene.">Surface-induced orientation control of CuPc molecules for the epitaxial growth of highly ordered organic crystals on graphene.</a> Journal of the American Chemical Society. 135 (9), 3680-3687 (2013).</li><li>Feng, X., Ma, T., Yamaura, D., Tadaki, D., Hirano-Iwata, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+characterization+of+air-stable+lipid+bilayer+membranes+incorporated+with+phthalocyanine+molecules.">Formation and characterization of air-stable lipid bilayer membranes incorporated with phthalocyanine molecules.</a> The Journal of Physical Chemistry B. 123 (30), 6515-6520 (2019).</li><li>Wu, Y., He, K., Ludtke, S. J., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+diffraction+study+of+lipid+bilayer+membranes+interacting+with+amphiphilic+helical+peptides:+diphytanoyl+phosphatidylcholine+with+alamethicin+at+low+concentrations.">X-ray diffraction study of lipid bilayer membranes interacting with amphiphilic helical peptides: diphytanoyl phosphatidylcholine with alamethicin at low concentrations.</a> Biophysical Journal. 68 (6), 2361-2369 (1995).</li><li>Zaitseva, S. V., Bettini, S., Valli, L., Kolker, A. M., Borovkov, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atypical+film-forming+behavior+of+soluble+tetra-3-nitro-substituted+copper+phthalocyanine.">Atypical film-forming behavior of soluble tetra-3-nitro-substituted copper phthalocyanine.</a> ChemPhysChem. 20 (3), 422-428 (2019).</li><li>Ghani, F., Gojzewski, H., Riegler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleation+and+growth+of+copper+phthalocyanine+aggregates+deposited+from+solution+on+planar+surfaces.">Nucleation and growth of copper phthalocyanine aggregates deposited from solution on planar surfaces.</a> Applied Surface Science. 351, 969-976 (2015).</li></ol>

In the precursor solution of the hybrid membrane, a mixed organic solvent (chloroform and hexane) rather than pure chloroform is used to dissolve lipids and CuPc. If pure chloroform is used, the density of the precursor solution would be higher than water. Therefore, it is highly likely that the solution would sink to the bottom of water rather than spread on water surface. Adding hexane, a low density solvent, to the precursor solution, ensures that the solution will float on the water surface and form a uniform hybrid ...

As essential frameworks of cell membranes, the interior of cells are separated from the exterior by a lipid bilayer system. This system consists of amphiphilic phospholipids, which are composed of hydrophilic phosphoric ester &#8220;heads&#8221; and hydrophobic fatty acids &#8220;tails&#8221;. Due to remarkable fluidity and self-assembly ability of lipid bilayers in aqueous environment1,2, artificial lipid bilayers can be formed using simple methods3,4. Various types of membrane proteins, such as ion channels, membrane receptors and enzymes, have been incorporated into the artificial lipid bilayer to mimic and study the functions of cell membranes5,6. More recently, lipid bilayers have been doped with nanomaterials (e.g., metal nanoparticles, graphene, and carbon nanotubes) to form functional hybrid membranes7,8,9,10,11,12,13. A widely used method for forming such hybrid membranes involves the formation of doped lipid vesicles, which contain hydrophobic materials such as modified Au-nanoparticles7 or carbon nanotubes11, and the resulting vesicles are then fused into planar supported lipid bilayers. However, this approach is complex and time-consuming, which limits the potential uses of such hybrid membranes.
In this work, lipid membranes were doped with organic molecules to produce hybrid lipid membranes that formed at the water/air interface by self-assembly. This protocol involves three steps: preparation of the mixed solution, formation of a hybrid membrane at the water/air interface, and the transfer of the membrane onto a Si substrate. Compared with other previously reported methods, the method described here is simpler and does not require sophisticated instrumentation. Using this method, air-stable hybrid lipid membranes with a larger area can be formed in a shorter time. The nanomaterial used in this study is a semiconducting organic molecule, copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc), which is widely used in a number of applications, including solar cells, photodetectors, gas sensors and catalysis14,15. CuPc, a small organic molecule with a planar structure, has a high affinity for the &#8220;tails&#8221; of phospholipids duo to its hydrophobic characteristics. Other groups have reported that CuPc molecules can self-assemble on single-crystal surfaces with the formation of highly ordered structures16,17. Therefore, it is highly possible that the CuPc molecules could be incorporated into the lipid bilayers through self-assembly.
We provide a detailed description of the procedures used to form membranes and provide some suggestions for smoothly implementing this procedure. In addition, we present some presentative results of the hybrid lipid membranes, and discuss potential applications of this method.

<table>
	<tbody>
		<tr>
			<td>Chloroform</td>
			<td>Wako&#160;Chemicals</td>
			<td>033-08631</td>
			<td></td>
		</tr>
		<tr>
			<td>CuPc</td>
			<td>Sigma-Aldrich</td>
			<td>423165</td>
			<td></td>
		</tr>
		<tr>
			<td>DPhPc</td>
			<td>Avanti Polar Lipids</td>
			<td>850356C</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials with screw cap</td>
			<td>Nichiden-Rike Glass Co., Ltd</td>
			<td>6-29801</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Wako&#160;Chemicals</td>
			<td>084-03421</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane filters</td>
			<td>Merck Millipore Ltd.</td>
			<td>R8CA42836</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-syringe</td>
			<td>Hamilton</td>
			<td>80530</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Tokyo Rikakikai Co., Ltd.</td>
			<td>11914199</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries, Inc.</td>
			<td>SI-0286</td>
			<td></td>
		</tr>
	</tbody>
</table>

self-assembly of hybrid lipid membranes doped with hydrophobic organic molecules at the water/air interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

The as-formed membrane has a uniform light blue color due to the presence of CuPc molecules. The area of the colored membrane is normally several square centimeters. In Figure 1A and Figure 1B, we show a microscopic image and an atomic force microscope (AFM) image (including a height profile) of the hybrid lipid membrane on a Si substrate. In the AFM image, the membrane in the upper left is thick, with a thickness of 79.4 nm and that at the lower right is thin, ...

Watch this Scientific Journal Video about Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface at JoVE.com

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

Our protocol demonstrate a simple method to fabricate organic molecule doped hybrid lipid membranes. The air stable property of the membrane can extend the application of lipid bilayer structure to solid state devices. We use a self-assembly process to form hybrid lipid membranes with a thickness of several nanometers.This protocol is simple and easy to follow and doesn't require complicated equipment. This method can be used to fabricate other same biohybrid lipid membranes and can be readily adopted to both sensors and other sensing devices. Working in an anaerobic glove box, dissolve copper phthalocyanine in chloroform inside a washed glass vial to prepare a 10 milligram per milliliter copper phthalocyanine stock solution.Filter the solution through a 0.2 micrometer PTFE membrane. Mix the DPHPC solution with a vortex mixer at 2, 300 RPM for 10 seconds. Then rinse a glass micro-syringe with chloroform five times and use it to transfer 200 microliters of the solution into a pre-washed glass vial.Evaporate the solvent in the vial with a gentle stream of nitrogen. Rinse another glass micro-syringe with chloroform, then use it to add 200, 2.6 microliters of chloroform to the glass vial with DPHPC. Add 47.4 microliters of the filtered copper phthalocyanine stock solution into the DPHPC solution which should result in a molar ratio of 10 to one DPHPC to phthalocyanine.Use another clean syringe to add 250 microliters of hexane to the solution. Then mix it with a vortex mixer at 2, 300 RPM for 10 seconds. Filter the prepared solution through a 0.2 micrometer PTFE membrane.Cut three by three centimeter silicon substrates from a silicon wafer. Then clean them in an ultrasonic bath for 10 minutes in purified water, followed by ethanol, and then chloroform. Treat the substrates with oxygen plasma for five minutes to remove adsorbed organic materials from the surface and to improve hydrophilicity.Wash a PTFE beaker with flowing purified water for three minutes. Then put the cleaned silicon substrate in the beaker tilted with a small angle. Pour a sufficient amount of purified water into the beaker until the entire silicon substrate is submerged.Take the prepared hybrid solution out of the freezer and allow it to warm to room temperature. Then stir it with a vortex mixer at 2, 300 RPM for 15 seconds. Use a rinsed 50 microliter micro-syringe to drop three to five microliters of the hybrid solution onto the water surface forming a floating hybrid lipid membrane.To transfer the membrane onto the silicon substrate, evaporate the organic solvent and pump the water out of the beaker with a peristaltic pump at a rate of three milliliters per minute. After the transfer process is complete, place the silicon substrate on a clean room wiper and allow all residual water to evaporate. The as formed hybrid lipid membrane has a uniform light blue color due to the presence of copper phthalocyanine molecules, and an area of several square centimeters.Shown here are confocal microscopy images and atomic force microscopy images of the membrane on a silicon substrate. In the AFM image, the membrane in the upper left is thick with a thickness of 79.4 nanometers, and that in the lower right is thin with a thickness of 4.9 nanometers. The thin membrane shows a surface roughness of 0.4 nanometers, which is close to that of the cleaned silicon substrate.Energy dispersive X-ray analysis was used to further investigate the composition of the hybrid membrane on the silicon substrate. The atomic ratios of representative elements, such as copper, phosphorous, nitrogen, and carbon are 0.33, 0.97, 4.06, and 68.56%respectively. The theoretical molar ratio of copper, to phosphorus, to nitrogen, to carbon should be one, to three, to 11, to 192, which is close to the measured element ratio in the hybrid membrane, indicating that the ratio between the lipids and the copper phthalocyanine molecules is maintained after the film formation and transfer processes.By doping the lipid membranes with other nanomaterials, such as graphene or menthol nanoparticles, nanohybrid membranes with various functions can be readily formed.

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Chemistry

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

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 ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

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 ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Synthesis and Characterization of Fe-doped Aluminosilicate Nanotubes with Enhanced Electron Conductive Properties

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe aims at lowering the band gap of imogolite, an insulator with the chemical formula (OH)3Al2O3SiOH, and at modifying its adsorption properties towards azo-dyes, an important class of organic pollutants of both wastewater and groundwater.
Fe-doped nanotubes are obtained in two ways: by direct synthesis, where FeCl3 is added to an aqueous mixture of the Si and Al precursors, and by post-synthesis loading, where preformed nanotubes are put in contact with a FeCl3&#8226;6H2O aqueous solution. In both synthesis methods, isomorphic substitution of Al3+ by Fe3+ occurs, preserving the nanotube structure. Isomorphic substitution is indeed limited to a mass fraction of ~1.0% Fe, since at a higher Fe content (i.e., a mass fraction of 1.4% Fe), Fe2O3 clusters form, especially when the loading procedure is adopted. The physicochemical properties of the materials are studied by means of X-ray powder diffraction (XRD), N2 sorption isotherms at -196 &#176;C, high resolution transmission electron microscopy (HRTEM), diffuse reflectance (DR) UV-Vis spectroscopy, and &#950;-potential measurements. The most relevant result is the possibility to replace Al3+ ions (located on the outer surface of the nanotubes) by post-synthesis loading on preformed imogolite without perturbing the delicate hydrolysis equilibria occurring during nanotube formation. During the loading procedure, an anionic exchange occurs, where Al3+ ions on the outer surface of the nanotubes are replaced by Fe3+ ions. In Fe-doped aluminosilicate nanotubes, isomorphic substitution of Al3+ by Fe3+ is found to affect the band gap of doped imogolite. Nonetheless, Fe3+ sites on the outer surface of nanotubes are able to coordinate organic moieties, like the azo-dye Acid Orange 7, through a ligand-displacement mechanism occurring in an aqueous solution.

Here, we present a protocol to synthesize and characterize Fe-doped aluminosilicate nanotubes. The materials are obtained by either sol-gel synthesis upon addition of FeCl3&#8226;6H2O to the mixture containing the Si and Al precursors or by post-synthesis ionic exchange of preformed aluminosilicate nanotubes.

The goal of the protocol is to synthesize Fe-doped aluminosilicate nanotubes of the imogolite type with the formula (OH)3Al2-xFexO3SiOH. Doping with Fe ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...