The overall goal of the following experiment is to create a mimic of the highly organized secondary cell wall of woody plants that have cellulose nano fis deposited in layers with lignin. This is achieved by oxidizing pulp fiber to create negatively charged carboxylate groups to allow fibrillation into nanocellulose and also assembly using layer by layer absorption as a second step, the nanocellulose is deposited into highly organized films, which are monitored using a quartz crystal micro balance with dissipation monitoring. Next nanocellulose is assembled onto dissolvable substrates in order to create freestanding films.
Results are obtained that show highly structured wood polymer films based on quartz crystal micro balance measurements, and microscopy measurements. The main advantage of this technique over existing methods like solvent casting a random mixture of the components is that you have control over the way the materials are organized within the film in order to mimic natural materials like wood. This method can help answer key questions in the cellulose by fields field, such as how do enzymes penetrate the highly structured cell wall of plants in order to convert cellulose into formable sugars?
To begin, set up a three liter, three neck flask with two liters of deionized water, an overhead stir, and a pH probe Add dignified craft pulp, 88%brightness tempo, and sodium bromide to the flask. As detailed in the text protocol, mix the pulp fiber with the overhead stir until the fiber is dispersed and no aggregates can be seen in the reaction. Initiate the oxidation by slowly adding a 12%solution of sodium hypochlorite to the reaction mixture through a syringe pump that delivers the sodium hypochlorite with an injection rate of 1.5 milliliters per minute.
Fill a second syringe with sodium hydroxide and manually meter the alkali solution into the flask dropwise to maintain the pH at 10 plus or minus 0.2. Monitor the change in pH with time. Once all the accessible hydroxyl groups on the cellulose are oxidized, the pH will no longer decrease and the reaction is complete.
Then add excess ethyl alcohol to consume the remaining sodium hypochlorite. Approximately six milliliters of 200 proof ethyl alcohol will consume all 100 milli mole of the original sodium hypochlorite. Filter and wash the oxidized fiber thoroughly with purified water to remove the reagents until the pH is neutral.
Then use a basket centrifuge or some filtration device like a bookner funnel to recover the fiber. Next, create a 3%slurry of the tempo oxidized pulp, and blend in a warring blender until the slurry becomes viscous and the blades start spinning in air because of gelling in the suspension, dilute the blended slurry to 0.1%and continue blending until the suspension becomes transparent. Prepare the aqueous solutions listed in the text protocol and adjust each solution with 0.1 molar sodium hydroxide to a pH of 10.5.
Clean a gold coated quartz crystal following the manufacturer's recommendation of using a base piranha solution for 10 minutes. Then rinse the crystals with purified water and blow dry them in a stream of nitrogen gas. Once dry, immediately insert the quartz crystal into the quartz crystal micro balance flow cell to avoid contamination from the air.
Pass the buffer through the flow cell to obtain a baseline response of the resonating crystal exposed to the liquid. Then deposit a layer of PDDA onto the quartz crystal by exposing the quartz crystal to the PDDA solution for five minutes. After five minutes, switch back to the buffer solution.
Repeat the absorption of other polymers in the sequence listed in the text protocol with a buffer rinse between each step. Repeat the cycle four times to deposit 16 total layers of polymers and nanoparticles. Glue a circular disc of MICA to a glass microscope slide using a quick epoxy adhesive.
After the adhesive cures, attach a piece of tape to the mica disc. Peel the tape away causing the Micah surface to cleave. Then dip the freshly cleaved mica that is attached to a glass slide in each prepared solution following the same sequence as before.
Image the deposited layers with an atomic force microscope. Use the intermittent contact mode and cantilevers with 10 nanometer radius. Silicon tips when collecting images of the sample set.
Scan size as 2.5 by 2.5 microns. Scan point as five 12 and integral gain of 10 to collect specific sample images for thickness measurements of the layers with a FM of the dried layer by layer films. Use a soft plastic pipette tip and scar a line across the surface of the prepared layer by layer films on the MICA surface.
To begin preparation of free standing layer by layer film cut a 25.4 millimeter by 7.6 millimeter rectangle of cellulose acetate film that is 0.13 millimeters thick and attached to an automated dipper arm. Then fill each 500 milliliter beaker with solutions of PDDA, lignin and nanocellulose according to concentration. And PH.Fill three additional beakers with aqueous buffer to use as a rinse solution for each deposition cycle.
Program the dipper arm to proceed in the same sequence as before. Change the solutions in the beaker during 250 cycles periodically as they begin to appear cloudy because of colloidal complexes. Following deposition, carefully trim the edges of the dried sample with scissors exposing the cellulose acetate edge and place into a covered glass petri dish filled with acetone to dissolve the cellulose acetate.
As a final step, soak isolated films in acetone for 24 hours. Rinse the films repeatedly with acetone to maximize the removal of residual cellulose acetate before analysis with scanning electron microscopy, the layer by layer absorption of lignin oxidized nano fial cellulose and PDDA was monitored in real time with QCMD to detect changes in frequency when molecules absorb to the surface of the quartz crystal. The data represents the normalized change in frequency and dissipation of the seventh overtone.
A baseline was first obtained with pH 10.5 MQ water, followed by introduction of the onic polymer PDDA. The introduction of this polymer is associated with a decrease in frequency and a corresponding increase in dissipation. This response is attributed to a combination of absorption of the PDDA on the gold courted quartz substrate and the change in the bulk effects of the liquid in contact with the vibrating crystal.
This was followed by a rinse step with the buffer to remove the excess unbound polymer and to negate the frequency of dissipation response due to the bulk effects of the polymer solution. The net change in frequency and dissipation from the baseline is due to the irreversible absorption of PDDA. Then the lignin solution was introduced, which resulted in a decrease in frequency and a corresponding increase in dissipation.
The crystal was then rinsed again, causing a slight increase in frequency. However, the dissipation remained unchanged, which suggests that lignin is deposited as a rigid layer over the PDDA layer when in contact with gold coated quartz substrate to deposit the second bilayer, the PDDA solution was reintroduced over the lignin layer. The introduction of PDDA solution was associated with a slight decrease in frequency and a significant increase in dissipation.
However, after the initial drop, there was a gradual increase in frequency followed by a plateau After the buffer rinse. The net change in frequency and dissipation after the deposition of PDDA on the lignin layer was found to be slightly lower than the previous layer. This change is the result of a strong interaction between PDDA and lignin, which may have caused partial desorption of loosely bound lignin.
The nanocellulose solution was introduced on the PDDA layer resulting in a decrease in frequency and a corresponding increase in dissipation. This change was found to be irreversible after the rinse step suggesting that NANOCELLULOSE had been irreversibly deposited on PDDA Atomic force Microscopy images show the lignin absorbed along the PDDA coated FIS after the lignin deposition step as seen in the images after three cycles in amplitude and height images, scanning electron microscopy of the cryo fractured cross sections of freestanding layer by layer films displays a lamellar structure. The thickness of two different types were found to be approximately 4.3 microns, which implies an average thickness of approximately 17 nanometers per deposition cycle.
While attempting this procedure, it's important to remember that oxidation of the P fiber is a key to the success of creating nanos. After watching this video, you should have a good understanding of how to isolate nanocellulose and use layer by layer assembly procedures to create materials with structured nanoscale architecture.
