Financial support from an Industrial Research Chair funded by FPInnovations and NSERC for a NSERC Discovery grant and from the NSERC Innovative Green Wood Fiber Products Network are acknowledged.

CAUTION: Read the material safety data sheets (MSDS) of all the chemicals before touching them. Many of the chemicals used in this work may cause severe health damages. Using personal protection such as lab coat, gloves, and goggles is a must. Do not forget that safety comes first. The water used throughout the synthesis is distilled water.
1. Preparation of Partially Oxidized Fibers as an Intermediate
<ol>
	<li>Tear 4 g Q-90 softwood pulp sheets into small pieces of approximately 2 x 2 cm2.</li>
	<li>Soak the torn pulp sheets in water for at least one day.</li>
	<li>Disintegrate the wet pulp using a mechanical disintegrator to achieve an almost-uniform dispersion.</li>
	<li>To assemble the vacuum filter, secure a nylon filter in a Buchner funnel and place the funnel in a filter flask. Then, connect the filter flask to a vacuum pump using proper tubing. Turn on the pump and pour the disintegrated pulp solution in the funnel to separate the pulp from the liquid.</li>
	<li>Measure the weight of wet pulp (m1), and calculate the amount of absorbed water by pulp: mw,1 = m1 - 4.</li>
	<li>Preparation of periodate oxidizing solution
	<ol>
		<li>For SNCC/DAMC synthesis: separately, dissolve 2.64 g sodium periodate (NaIO4) and 15.48 g sodium chloride (NaCl) in 200-mw,1 ml water.</li>
		<li>For ENCC/DCC synthesis: separately, dissolve 5.33 g sodium periodate (NaIO4) and 15.6 g sodium chloride (NaCl) in 266-mw,1 ml water.</li>
	</ol>
	</li>
	<li>Add the wet pulp separately to the solutions prepared in 1.6. Make sure that the total amount of water (absorbed by pulp plus added water) is equal to 200 ml for SNCC and 266 ml for ENCC syntheses.</li>
	<li>Cover the beaker thoroughly with aluminum foil to prevent periodate deactivation while stirring at speed ~ 105 rpm in RT for a desired amount of time according to Table 1 to achieve a favored aldehyde content. As an example, to obtain ~ 6.5 mmol/g aldehyde, react for 96 hr.</li>
	<li>When the reaction time is elapsed, open the aluminum foil and add 1 ml (in case of SNCC/DAMC synthesis) or 3 ml (in case of ENCC/DCC synthesis) ethylene glycol to the mixture and stir for 10 min to stop the oxidation reaction by quenching periodate.</li>
	<li>Collect the oxidized pulp by vacuum filtration (according to 1.4), redisperse it in 500 ml water, and stir it for 30 min. Repeat this step at least 5 times to clean the pulp from periodate thoroughly.</li>
	<li>After the 5th water wash on the oxidized pulp, separate the pulp from solution by vacuum filtration and store it in a cold (4 &#176;C) place.</li>
</ol>
2. Synthesis of SNCC and DAMC
<ol>
	<li>Divide the partially oxidized wet pulp (m1), obtained in 1.11, by four: m2 = m1/4, and measure the weight of absorbed water: mw,2 = m2 - 1.</li>
	<li>Disperse the pulp in (100 - mw,2) g water in a round bottom flask (total water content = 100 g).</li>
	<li>Place the round bottom flask in an oil bath and heat the partially oxidized pulp at 80 &#176;C for 6 hr while gently stirring. 
	Note: If pulp is completely oxidized with periodate (DS = 2), e.g., by reacting 1 g pulp with 1.85 g NaIO4 (8.65 mmol) in a solution comprising 3.87 g NaCl (8.64 mmol) and 65 ml water while stirring for 6 days, depending on the heating condition and residence time in water, the property of the dialdehyde cellulose (DAC) is changed (Table 2).</li>
	<li>Cool down the solution to RT.</li>
	<li>Centrifuge the solution at 18,500 x g for 10 min. The precipitate is unfibrillated cellulose (fraction 1).</li>
	<li>Separate the supernatant carefully and weigh it (A).</li>
	<li>Add 1.7(A) g propanol to the supernatant obtained in 2.6 while stirring to precipitate SNCC. Details about the separated SNCC and added propanol is available in Figure 1.</li>
	<li>Centrifuge the biphasic solution at 3,000 x g for 10 min, and separate the resulted gel-like precipitate (second fraction, SNCC) by decantation, which is ready to be redispersed and dialyzed for further purification (section 4) and characterization (section 5).</li>
	<li>To the supernatant obtained in 2.8, add 3.5(A) g propanol to yield a white precipitate (third fraction, DAMC).</li>
	<li>Centrifuge the solution of 2.9 at 3,000 x g for 10 min, and collect the gel-like DAMC precipitate (by pouring the supernatant in a separate beaker) ready to be redispersed in water, purified by dialysis (details available in section 4), and characterized (section 5).</li>
</ol>
3. Synthesis of ENCC and DCC
<ol>
	<li>Prepare a solution of 0.5 M sodium hydroxide (NaOH) by dissolving ~ 2 g NaOH in 100 ml water and keep it aside. This will be used in step 3.7.</li>
	<li>Divide the wet oxidized pulp, obtained in 1.11, by four: m3 = m1/4, and measure the weight of absorbed water: mw,3 = m3 - 1.</li>
	<li>Separately, add 2.93 g sodium chloride (NaCl) and 1.41 sodium chlorite (NaClO2) to (50 - mw,3) ml water and stir to dissolve.</li>
	<li>Suspend m3 gram of wet oxidized pulp (containing ~ 1 g dry oxidized pulp) in the solution obtained in 3.3. Note that the final pulp concentration is 1 g in 50 ml total available water (free and absorbed water).</li>
	<li>Place a pH meter in the solution of 3.4.</li>
	<li>Add 1.41 g hydrogen peroxide (H2O2) to the mixture of step 3.4 dropwise.</li>
	<li>Stir the suspension of 3.6 for 24 hr in RT at 105 rpm while maintaining the pH ~ 5 by gradually adding 0.5 M sodium hydroxide (NaOH) prepared in step 3.1. 
	Note: The pH starts decreasing rapidly after ~ 15 min from the beginning of reaction, and it should be kept constant at 5 for at least the first 4 hr of the reaction. For convenience, it is suggested that the reaction is started at 1 PM and the pH is controlled until 5 PM, then the reaction is left O/N and early in the morning the pH is increased to 5 again. After such a long time, pH drop will not be significant, indicating that most of the conversion is achieved. Now, almost no solid can be observed in the solution (large fibers are broken down into nanoparticles). Note that if the reaction is left for a longer time, the crystalline part may be disrupted.</li>
	<li>Divide the suspension obtained from 3.7 into equally weighted centrifuge tubes and centrifuge at 27,000 x g for 10 min, and separate the supernatant (ENCC + DCC) from the micro-fibrous precipitate.</li>
	<li>Weigh the supernatant obtained from 3.8 and call the solution mass (B).</li>
	<li>Slowly add 0.16(B) g ethanol to the solution of 3.9 while stirring to form a white precipitate (second fraction, ENCC).</li>
	<li>Centrifuge the solution of 3.10 at 3,000 x g for 10 min, and separate the resulted gel-like ENCC precipitate by decantation. ENCC is ready to be redispersed in water, purified by dialysis (details available in section 4), and characterized (section 5).</li>
	<li>To the supernatant obtained in 3.11, add equal mass of ethanol as the solution mass to yield a white precipitate (third fraction, DCC).</li>
	<li>Centrifuge the solution of 3.12 at 3,000 x g for 10 min, and separate the gel-like DCC precipitate ready to be redispersed in water, purified by dialysis (details available in section 4), and characterized.</li>
</ol>
4. Dialysis Procedure to Purify SNCC, DAMC, ENCC or DCC
<ol>
	<li>Redisperse the gel-like precipitate obtained in any steps of 2.8 (SNCC), 2.10 (DAMC), 3.11 (ENCC), or 3.13 (DCC) in 10 ml water by vigorous stirring for 1 hr.</li>
	<li>Place the dispersion in a dialysis tubing (MW Cutoff = 12-14 kDa, Length ~ 30 cm, width ~ 4.5 cm) and secure the top and bottom by clipping.</li>
	<li>Place the filled dialysis bag in ~ 4 L of distilled water and stir for 24 hr to eject the salts.</li>
	<li>Collect the dialyzed solution in a container and store in a cold (4 &#176;C) place.</li>
</ol>
5. Post-purification Characterization: Solid Phase and Charge Concentrations Measurement
<ol>
	<li>Concentration measurement
	<ol>
		<li>Weigh 3 ml of a desired dispersion in a weighting dish (Aluminum cup, 57 mm).</li>
		<li>Place the weighing dish containing the dispersion in an oven (50 &#176;C) O/N.</li>
		<li>Weigh the dry film and calculate the concentration of nanoparticles or polymers in the dispersion: 
		Concentration (w/v%) = 100 x mass of dry film / 3, or 
		Concentration (w/w%) = 100 x mass of dry film / mass of dispersion</li>
	</ol>
	</li>
	<li>Conductometric titration
	<ol>
		<li>Conductometric titration of SNCC or DAMC to determine aldehyde content
		<ol>
			<li>Prepare 0.1 M hydrochloric acid (HCl) by adding 0.82 ml HCl to 25 ml water followed by adjusting the final volume to 100 ml.</li>
			<li>Separately, prepare NaOH 0.1 M by adding 0.4 g sodium hydroxide to distilled water to achieve 100 ml final solution.</li>
			<li>Following the hydroxylamine hydrochloride method24, add a known amount of a desired dispersion to a desired amount of water (e.g., 0.02 g in 50 ml H2O).</li>
			<li>Adjust the pH to 3.5 using dilute HCl (0.1 M).</li>
			<li>Add 10 ml hydroxylamine hydrochloride solution (5% w/w) to the dispersion.</li>
			<li>Monitor the pH and keep it at 3.5 by adding 0.1 M NaOH until pH becomes stable at 3.5.</li>
			<li>Using the consumed volume of NaOH to neutralize the H+ released from the reaction of aldehyde groups and NH2OH&#183;HCl, measure the aldehyde concentration (mole of consumed NaOH = mole of produced HCl during the reaction = mole of aldehyde groups on SNCC).</li>
		</ol>
		</li>
		<li>Conductometric titration of ENCC or DCC to determine carboxyl content
		<ol>
			<li>Following literature25, add enough amount of a desired dispersion to have 0.02 g of solid in 140 ml distilled water.</li>
			<li>Separately, prepare 20 mM NaCl by dissolving 0.117 g NaCl in distilled water to achieve 100 ml final solution. Add 2 ml of 20 mM NaCl to 5.2.2.1.</li>
			<li>Reduce the pH to about 3 using dilute HCl (0.1 M).</li>
			<li>Perform the conductometric titration by adding standard sodium hydroxide (NaOH, 10 mM) in 0.1 ml/min increments up to pH ~ 11.</li>
			<li>Using the consumed volume of NaOH to neutralize charged groups (details in Figure 2), measure the surface charge concentration (1 mole of consumed base equals to one mole COOH on the particle surface).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Following the chemistry discussed in this visual paper, a spectrum of highly stable cellulose-based nanoparticles with tunable charge bearing both crystalline and amorphous phases (hairy nanocrystalline celluloses) are produced. Depending on the periodate oxidation time, as shown in Table 1, various products are yielded: oxidized fibers (fraction 1), SNCC (fraction 2), and DAMC (fraction 3) each of which providing unique properties, such as defined size, morphology, crystallinity, and aldehyde content. F...

Cellulose, as the most abundant biopolymer in the world, has been served recently as a key raw material to yield crystalline nanoparticles named nanocrystalline cellulose (NCC, also known as cellulose nanocrystals CNC)1. To understand the mechanism of NCC synthesis, the structure of cellulose fibers needs to be explored. Cellulose is a linear and polydispersed polymer comprising poly-beta(1,4)-D-glucose residues2. The sugar rings in each monomer are connected through glycosidic oxygen to form chains of (1-1.5) x 104 glucopyranose units2,3, introducing alternating crystalline parts and disordered, amorphous regions, first reported by Nageli and Schwendener2,4. Depending on the source, crystalline parts of cellulose can adopt various polymorphs5.
If a cellulose fiber is treated with a strong acid, such as sulfuric acid, the amorphous phase can be completely hydrolyzed away to disrupt the polymer and produce crystalline particles of various aspect ratio depending on the source (e.g., wood and cotton yield more than 90% crystalline nanorods of width ~ 5-10 nm and length ~ 100-300 nm, whereas tunicin, bacteria, and algae produce 5-60 nm wide and 100 nm to several micrometer long NCCs)6. Readers are referred to the vast amount of literature available on the scientific and engineering aspects of these nanomaterials2,5,7-16. Despite numerous interesting properties of these nanoparticles, their colloidal stability has always been an issue at high salt concentrations and high/low pH due to their relatively low surface charge content (less than 1 mmol/g)17.
Instead of strong acid hydrolysis, cellulose fibers can be treated with an oxidizing agent (periodate), cleaving C2-C3 linkage in the anhydro D-glucopyranose residues to form 2,3-dialdehyde units with no significant side reactions18,19. These partially oxidized fibers can be used as a valuable intermediate material to produce nanoparticles bearing both amorphous and crystalline regions (hairy nanocrystalline celluloses) using solely chemical reactions without any mechanical shear or ultrasonication20. When the partial oxidation degree DS &#60; 2, heating oxidized fibers results in three batches of products, namely fibrous cellulose, water dispersible dialdehyde cellulose nanowhiskers called sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which can be isolated by precise control over the co-solvent addition and intermittent centrifugation21.
Performing controlled chlorite oxidation on the partially oxidized fibers converts almost all the aldehyde groups to carboxyl units, which can introduce as high as 7 mmol COOH groups per gram of nanocrystalline cellulose depending on the aldehyde content18, acting as stabilizers. These nanoparticles are called electrosterically stabilized nanocrystalline cellulose (ENCC). Furthermore, it has been confirmed that soft layers of charged hair-like protruding chains exist on ENCC17. This material has been used as a highly efficient adsorbent to scavenge heavy metal ions22. The charge of these nanoparticles can be precisely controlled by controlling the periodate reaction time23.
Despite known oxidation reactions of cellulose, the production of SNCC and ENCC has never been reported by any other research groups most probably due to the separation challenges. We have been able to successfully synthesize and isolate various fractions of nanoproducts by precisely designing the reaction and separation steps. This visual article demonstrates with complete detail how to reproducibly prepare and characterize the aforementioned novel nanowhiskers bearing both amorphous and crystalline parts from wood fibers. This tutorial may be an asset for active researchers in the fields of soft material, biological, and medicinal sciences, nanotechnology and nanophotonics, environmental science and engineering, and physics.

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Q-90 softwood pulp</td>
			<td style="width:149px;">FPInnovations</td>
			<td style="width:213px;">-</td>
			<td style="width:369px;">-</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium periodate</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:213px;">S1878-500G/CAS7790-28-5</td>
			<td style="width:369px;">Light sensitive, Strong oxidizer, must be kept away from flammable materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:149px;">ACP Chemicals</td>
			<td style="width:213px;">S2830-3kg/7647-14-5</td>
			<td style="width:369px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-Propanol</td>
			<td style="width:149px;">Fisher</td>
			<td style="width:213px;">L-13597/67-63-0</td>
			<td style="width:369px;">Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:213px;">102466-1L/107-21-1</td>
			<td style="width:369px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:149px;">Fisher</td>
			<td style="width:213px;">L-19234/1310-73-2</td>
			<td style="width:369px;">Strong base, causes serious health effects</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chlorite</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:213px;">71388-250G/7758-19-2</td>
			<td style="width:369px;">Reactive with reducing agents and combustible materials</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydrogen peroxide</td>
			<td style="width:149px;">Fisher</td>
			<td style="width:213px;">H325-500/7722-84-1</td>
			<td style="width:369px;">Corrosive and oxidizing agent, keep in a cool and dark place</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:149px;">Commercial alcohols</td>
			<td style="width:213px;">P016EAAN</td>
			<td style="width:369px;">Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrochloric acid</td>
			<td style="width:149px;">ACP Chemicals</td>
			<td style="width:213px;">H-6100-500mL/7647-01-0</td>
			<td style="width:369px;">Strong acid, causes serious health effects</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydroxylamine hydrochloride</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:213px;">159417-100G/5470-11-1</td>
			<td style="width:369px;">Unstable at high temperature and humidity, mutagenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:213px;">J2</td>
			<td>High rotary speed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fixed angle rotor</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:213px;">JA-25.50</td>
			<td>Tighten the lid carefully</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Dialysis tubing</td>
			<td style="width:149px;">Spectrum Labs</td>
			<td style="width:213px;">Spectra (Part No. 132676)</td>
			<td>Cutoff Mw = 12-14 kD, Length ~ 30 cm, width ~ 4.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aluminum cup</td>
			<td style="width:149px;">VWR</td>
			<td style="width:213px;">611-1371</td>
			<td>57 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Titrator</td>
			<td style="width:149px;">Metrohm</td>
			<td style="width:213px;">836 Titrando</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

highly stable, functional hairy nanoparticles and biopolymers from wood fibers: towards sustainable nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

The mass portion and charge content of each fraction during the periodate and chlorite oxidation of pulp depends on the reaction time (Table 1). Moreover, DAC molecular weight depends on heating condition and residence time (Table 2). Once SNCC and DAMC are made, they precipitate out by adding propanol (Figure 1). To measure the charge content of ENCC, conductometric titration is performed (Figure 2). NCC and ENCC colloid...

Watch this Scientific Journal Video about Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology at JoVE.com

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

highly-stable-functional-hairy-nanoparticles-biopolymers-from-wood

Research

JoVE Journal

Chemistry

11.9K Views.  McGill University. Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed. 

Video: Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

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 ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Green and Low-cost Production of Thermally Stable and Carboxylated Cellulose Nanocrystals and Nanofibrils Using Highly Recyclable Dicarboxylic Acids

Here we demonstrate potentially low cost and green productions of high thermally stable and carboxylated cellulose nanocrystals (CNCs) and nanofibrils (CNF) from bleached eucalyptus pulp (BEP) and unbleached mixed hardwood kraft pulp (UMHP) fibers using highly recyclable dicarboxylic solid acids. Typical operating conditions were acid concentrations of 50 - 70 wt% at 100 &#176;C for 60 min and 120 &#176;C (no boiling at atmospheric pressure) for 120 min, for BEP and UMHP, respectively. The resultant CNCs have a higher thermal degradation temperature than their corresponding feed fibers and carboxylic acid group content from 0.2 - 0.4 mmol/g. The low strength (high pKa of 1.0 - 3.0) of organic acids also resulted in CNCs with both longer lengths of approximately 239 - 336 nm and higher crystallinity than CNCs produced using mineral acids. Cellulose loss to sugar was minimal. Fibrous cellulosic solid residue (FCSR) from the dicarboxylic acid hydrolysis was used to produce carboxylated CNFs through subsequent mechanical fibrillation with low energy input.

Here we demonstrate a novel method for green and sustainable productions of highly thermally stable and carboxylated cellulose nanocrystals (CNC) and nanofibrils (CNF) using highly recyclable solid dicarboxylic acids.

Here we demonstrate potentially low cost and green productions of high thermally stable and carboxylated cellulose nanocrystals (CNCs) and nanofibrils ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...