Name: using vertically aligned carbon nanofiber arrays on rigid or flexible substrates for delivery of biomolecules and dyes to plants
Uploaded: 2023-07-21T13:00:00.000+00:00
Duration: 5 min 32 s
Description: Watch this Scientific Journal Video about Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays at JoVE.com

Nanofiber arrays were fabricated at the Center for Nanophase Materials Sciences, which is a Department of Energy Office of Science User Facility (Proposal ID: CNMS2019-103 and CNMS2022-A-1182). Support from CNMS is awarded through a peer-reviewed proposal system and is provided at no cost to successful applicants who intend to publish their results (http://www.cnms.ornl.gov/user/becoming_a_user.shtml). We thank Kevin Lester and CNMS for assistance with the production of nanofiber arrays. We thank Dr. John Caughmen, Dr. Timothy McKnight, Dr. Amber Webb, Daryl Briggs, and Travis Bee for critical discussions on experimental design. We thank Dr. Adam Rondinone for the schematic of the PECVD machine. We thank Leslie Carol for the scientific illustrations. This work was funded by the Bioimaging Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, DE-SC0019104, and United States Department of Agriculture, 2021-67013-34835. JMM was supported by the United States Department of Agriculture: National Institute of Food and Agriculture: Agriculture and Food Research Initiative Predoctoral Fellowship 2021-67034-35167.

1. VACNFs production (Figure 2 and Figure 3)
<ol>
	<li>Spincoat a silicon wafer with polymethyl methacrylate (PMMA) 495 A4&#160;resist at 4000 rpm and bake it at 180 &#176;C for 5 min. 
	NOTE: Let the wafer cool for 10 s before proceeding to the next step.</li>
	<li>Spincoat a second layer of resist (PMMA 950 A2) and bake it at 180 &#176;C for 5 min.</li>
	<li>Use electron beam lithography to define the catalyst dots with diameters of 300 nm at a specified lateral spacing (pitch: 10 &#181;m, or 35 &#181;m) in 3 mm x 3 mm arrays.</li>
	<li>Develop the resist in 30-40 mL of 1:3 methyl-isobutyl ketone: isopropyl alcohol (IPA) for 1.5 min, followed by rinsing it with IPA and drying it with N2. 
	NOTE: Check for the array of dots using a brightfield microscope (20x objective).</li>
	<li>Clean the wafer of the residual resist with a 6 s exposure in oxygen plasma (descum) in a silicon etcher.</li>
	<li>Use electron beam evaporation to deposit first an adhesive layer of metal (Ti or Cr, 10 nm) and then deposit a second layer, which is the Ni catalyst (130 nm or 150 nm). During Ti or Ni metal deposition, keep the current below 0.2 A at 10 kV, the pressure below 5 x 10-6 Torr, and the deposition rate at ~1 A/s for line-of-sight deposition.</li>
	<li>Use sequential bath sonication to remove the metal (Ni) deposited on the underlying resist layer, leaving the Ni deposited directly on the silicon wafer. This process is called lift-off.
	<ol>
		<li>For this, prepare 3 containers with acetone and bath sonicate the wafer in acetone for 1 min; repeat 3 times at room temperature (RT, 20 &#176;C) at a frequency of 35 kHz. Rinse with IPA and dry with N2. 
		NOTE: Never let acetone dry on the wafer. After the last acetone sonication, immediately rinse with IPA and then dry with N2 gas. If at any point the wafer becomes prematurely dry (prior to IPA rinse) there is the possibility that not all the excess metal and resist will be removed and the acetone can leave a residue.</li>
	</ol>
	</li>
	<li>Check the geometry of catalyst shape using scanning electron microscopy (SEM). To capture SEM images of the VACNF chips, tilt the stage to a 30&#176; angle and use a voltage of 1-3 kV, with a beam current of ~100 pA, and a ~5 mm working distance. 
	NOTE: The catalyst should look like a hockey puck (a compact cylinder) (Figure 4). The height of the compact cylinder should reflect the thickness of Ni deposited on the silicon wafer. If the profile of the Ni dot is tall and appears to be concave at the top, resembling a volcano (Figure 4), then the catalyst is more likely to dewet into multiple Ni droplets resulting in branching of the carbon nanofibers9(Figure 4 and Figure 5). Such problems can result from resist melting during metal evaporation because of long deposition times or issues with the lift-off procedure (Figure 4 and Figure 5).</li>
	<li>Quarter the silicon wafer and place it in the direct current plasma enhanced chemical vapor deposition chamber (dc-PECVD) with the acetylene/ammonia mixture. Optimize the growth parameters to control the length and taper of nanofibers (tips &#60;200 nm).
	<ol>
		<li>Use a current of 1-1.5 A and a voltage range of 440-560 V. When growing the fibers, use a pre-treatment phase with ammonia flow while the machine and substrate heat up to 620 &#176;C. Ensure to turn on the carbon source (acetylene) 10 s before starting the plasma. 
		NOTE: Quarter wafers are used in case the PECVD machine run parameters need to be optimized. To produce fibers with lengths greater than 40 &#181;m and tips less than 200 nm in diameter, the following parameters are suggested: with a Ni catalyst thickness of 130 nm, use a current of 1.75 A, growth time of 70 min, and an acetylene : ammonia ratio of 45 standard cubic centimeter per minute (sccm) : 100 sccm. With a Ni thickness of 150 nm, use a current of 1.5 A, a growth time of 80 min, and an acetylene : ammonia ratio of 48 sccm : 100 sccm. Use the following parameters to grow VACNFs regardless of catalyst thickness: growth temperature of 620 &#176;C, pressure of 10 torr during plasma, and a shower head height of 20 mm (Figure 6).</li>
	</ol>
	</li>
	<li>Assess the geometry of the resulting fibers using SEM at a 30&#176; tilt with an acceleration potential of 1 kV. 
	NOTE: Optimal fibers will be straight and non-branching. It is impossible to control the crystal orientation of the Ni catalyst with the electron beam evaporator. As a result, there will be some fibers that branch because of catalyst dewetting9. Also, the crystal orientation causes the VACNFs to grow to different heights, so uniform heights on all VACNFs are difficult.</li>
	<li>Spincoat a layer of photoresist (SPR955) onto the fibers at 1000 rpm for 45 s. Then dice the &#188; wafer into 3 mm x 3 mm arrays using a dicing saw. Store the fibers at this point for later use. 
	&#8203;NOTE: Do not conduct step 1.11 if planning to transfer fibers to a flexible substrate.</li>
</ol>
2. Transferring VACNFs to a flexible substrate (Figure&#160;7 and Figure 8)
<ol>
	<li>After the fibers are synthesized, spincoat SU-8 2015 photoresist onto the wafers or quarter wafers at 4000 rpm for 45 s.</li>
	<li>Softbake the wafer for 3 min at 95 &#176;C.</li>
	<li>Using a contact aligner, expose the wafer with a 3 mm 3 mm array patterned mask that aligns with the defined pattern from electron beam lithography at 95 mJ/cm2. 
	NOTE: Use a proximity contact mode with an exposure gap that is at 20 &#181;m greater than the tallest fiber. There is the possibility that fibers could be knocked over during this step in the process.</li>
	<li>Perform a post-exposure bake for 6 min at 95 &#176;C.</li>
	<li>Develop the wafer in SU-8 developer for 15 s, rinse it with IPA, and dry the wafer with N2, moving from top to bottom. 
	&#8203;NOTE: When developing the wafer, ensure it is completely submerged.</li>
	<li>Spincoat patterned wafers with a protective layer of thin photoresist (SPR 955 CM 0.7) at 3000 rpm for 45 s and softbake for 30 s at 90 &#176;C.</li>
	<li>Deposit a thin silicon oxide layer to the wafer (2-3 nm) by placing it in an atomic layer deposition for 22 cycles at 100 &#176;C.</li>
	<li>Use a dicing saw to cut the wafer into 3 mm x 3 mm squares. Align the dicing saw to the preexisting pattern on the wafer.</li>
	<li>Assess the geometry of the resulting fibers using SEM at a 30&#176; tilt with an acceleration potential of 3 kV.</li>
	<li>Stop here if storing chips for long-term use (&#62;1 week). Store chips in the dark.</li>
	<li>To separate flexible substrates from rigid substrates, place individual chips in acetone for 30 min or until the SU-8 starts to curl.</li>
	<li>Wash SU-8 films (either attached to or detached from rigid substrates) with IPA for 5 min and then with water for 5 min. Place the chips in a commercial box with a sticky pad when transporting them.</li>
	<li>OPTIONAL: Place the fiberless side of the SU-8 film on water-soluble tape or on top of thin silicone rubber with a thin polyethylene terephthalate (PET) (12.5 &#181;m thick) backing.
	<ol>
		<li>Use a pair of tweezers to transfer the SU-8 film. Press on the edges of the SU-8 square to help it stick to the tape/silicone rubber-PET; this is to avoid breaking off fibers. At this point, the VACNF films are ready for immediate use.</li>
		<li>To prepare a silicone rubber/PET holder, do the following: using a 2-part kit for silicone rubber, mix the two parts together (the elastomer and the crosslinker). Next, cut out a square of PET and tape it down in a clear plastic dish. Pour a very thin layer of silicone rubber on top of the PET and cure it at 80 &#176;C for 1-2 h.</li>
	</ol>
	</li>
</ol>
3. On-plant method (where a drop of solution to deliver is placed on a plant surface) using fibers in a rigid substrate (Figure 1A)
<ol>
	<li>Remove the photoresist with increment washes of acetone (100%, 5 min), IPA (100%, 5 min), and ddH2O (5 min) prior to use.</li>
	<li>Place the plant tissue to be impaled on a hard surface for support.</li>
	<li>Place a 1 &#181;L droplet of dye or DNA&#160;(200 ng) onto the surface of the plant tissue.</li>
	<li>Place a VACNF chip with a rigid substrate on the top of the droplet, with the fibers oriented such that they will come into contact with the droplet. 
	NOTE: The orientation of the chips can be determined by the &#34;shininess&#34; of the wafer. The shiny side of the chip has fibers, and the opaque side does not.</li>
	<li>Using the flat side of a pair of tweezers, tap the chip. Mark the area of the plant that the chip came into contact with using a soft-tipped marker. Remove the VACNF chips after delivery. 
	NOTE: The amount of force applied when tapping will vary depending on the type of plant tissue used. It is recommended to practice tapping chips prior to conducting the impalement of fibers. Avoid damage to plant tissues. Damage is apparent when one can see the outline of the VACNF chip in the plant tissue.</li>
	<li>Repeat steps 3.1-3.5 for controls (+Dye/DNA, -Fibers; -Dye/DNA, +Fibers; and -Dye/DNA, -Fibers). -Fibers is the fiberless side of a chip.</li>
	<li>Store intact plants or excised plant organs in humid chambers in long-day conditions (16 h light, 8 h dark) if needed. For excised organs, use a plastic Petri dish with wet paper towels.</li>
</ol>
4. On-chip method (where a drop of solution to deliver is placed on a VACNF chip), rigid substrate (Figure 1B)
<ol>
	<li>Remove the photoresist with increment washes of acetone (100%, 5 min), IPA (100%, 5 min), and ddH2O (5 min) prior to use.</li>
	<li>Drop cast 1 &#181;L droplet of dye or plasmid DNA (200 ng) onto the fiber side of VACNF chip with rigid substrate. Ensure to place the droplet in the center of the chip and cover several fibers. Let the droplet dry for 15 min. 
	NOTE: The orientation of the chips can be determined by the &#34;shininess&#34; of the wafer. The shiny side of the chip has fibers, and the opaque side does not.</li>
	<li>When working with leaves or other excised organs, place them on top of a hard surface. When working with intact plants, place a hard surface beneath the organ to which VACNFs are applied.</li>
	<li>After the 15 min drying step, position the VACNF chip such that the fiber side comes in contact with the plant tissue. Tap the chip with the back end of a pair of tweezers. 
	NOTE: The amount of force applied when tapping will vary depending on the type of plant tissue used. It is recommended to practice tapping chips.</li>
	<li>Repeat steps 3.6-3.7 of the on-plant method.</li>
</ol>
5. Applying VACNFs in SU-8 films to plant tissue using the on-chip method (Figure 1C)
<ol>
	<li>Place 1 &#181;L droplet of dye or DNA (200 ng) onto the fiber side of the SU-8 film and let it dry for 10 min. Be sure to place the droplet in the center of the chip. 
	NOTE: There are different drying times depending upon the substrate used.</li>
	<li>Using a pair of sharp tweezers, place the VACNF film onto the plant surface. 
	NOTE: The longer the SU-8 films are left out in the air, the more brittle the films become. To limit the risk of losing the SU-8 films, ensure to have all plants/samples and equipment near the SU-8 films.</li>
	<li>Gently roll a small makeup applicator over the VACNF film. Mark the areas where the flexible substrates are placed with a soft-tipped marker. Remove the flexible substrates from the plant surface using tape. 
	NOTE: The amount of force applied when rolling the makeup applicator will vary among the plant tissues used. Practice applying the VACNF films prior to conducting biomolecule or dye delivery. Visible damage to plant tissues is apparent when one can see the outlines of VACNF film in the plant tissue.</li>
	<li>Repeat steps 5.1-5.3 for controls and store plants as mentioned in step 3.7 of the on-plant method.</li>
</ol>
6. Microscopy and image analysis for all delivery methods
<ol>
	<li>Image the samples using a confocal microscope using emission and excitation wavelengths suitable for the delivered fluorescent probe/reporter. 
	NOTE: The time required for the transient transformation varies with the plant species and the marker delivered. For example, the expression of fluorescent markers was detected after 48 h in Arabidopsis versus 96 h in lettuce leaves7.</li>
	<li>When imaging, try to focus on a region with detached fibers. Fibers will have different orientations. The success of delivery is not dependent on appearance of broken-off fibers. 
	NOTE: Fibers will be fluorescent with the most common excitation/emission settings due to the formation of silicon nitride layer resulting from the fiber formation in PECVD18.</li>
	<li>Capture at least 5 images for each sample. The resulting signal will vary.</li>
	<li>Measure the fluorescence values as total fluorescence (integrated density) in 20 &#181;m x 20 &#181;m confocal image areas7 using ImageJ19.</li>
</ol>

Using Carbon Nanofiber Arrays for Efficient Biomolecule and Dye Delivery to Plants

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The authors have nothing to disclose.

In this paper, we presented methods for constructing vertically aligned carbon nanofiber arrays, transferring the fibers to a flexible substrate, and applying fibers in either a rigid or flexible substrate to plants for use in the delivery of biomolecules or dyes to plants. We described two general approaches, the on-chip and the on-plant methods, for deposition of the introduced materials and showed successful results in fibers on a rigid substrate as well as the on-chip method using VACNF films. The application of these fibers is simpler in practice and theory than traditional plant transformation methods (particle bombardment, protoplast transformation via PEG or electroporation) and may be used for plants recalcitrant to Agrobacterium-mediated transformation. However, only a few cells are transformed.
Vertically aligned carbon nanofibers were produced at Oak Ridge National Laboratory Center for Nanophase Materials Sciences through their user program. Users can apply to use this facility for the production of VACNFs. Alternatively, VACNF chips can be produced in clean rooms with direct current plasma-enhanced chemical vapor deposition machines with a carbon source22,23. With the methods described, there are a few steps that are critical to the production of the fibers, fiber transfer, and application of the VACNF chips/films. For fiber application to work, fibers must be straight and have a tapering diameter of &#60;200 nm at the tip for delivery in plant cells to be successful6,7(Figure 3). To create carbon nanofibers of particular size and pitch, there are a variety of parameters that can be changed, including dot size, lateral pitch, and the amount of catalyst deposited. To select the optimal dot size to be used for the carbon nanofiber production, fibers were grown from various dot sizes (as shown in Figure 5). We found that 300 nm diameters produced the best fibers, hence this dot size was selected (Figure 5). After finding the right parameters, we looked to use chips that have &#62;50% of fibers with the ideal geometry (straight and a tip diameter &#60;200 nm). To check the geometry of the fibers, we used scanning electron microscopy to image random fields of view on a sample of VACNF chips/films.
Additionally, the fibers must have a certain minimum length to achieve delivery within plant cells. The importance of producing fibers of different length is that longer fibers could be used to penetrate deeper tissue layers. Longer fibers (&#62;40 &#181;m in length) are essential for flexible films as the fiber transfer works by breaking the fibers from their base and requires layering SU-8 on top of the fibers. The working thickness of the SU-8 layer used for this protocol is 20-35 &#181;m. The minimum height necessary to accomplish delivery within the epidermis of various plants (curved or flat) is 10-15 &#181;m6,7. As a result, fibers with lengths &#62;40 &#181;m are necessary for VACNF films. There are several different parameters to consider when producing carbon nanofibers: catalyst material, catalyst geometry, thickness of catalyst material as well as conditions within the PECVD chamber (gas ratio, pressure, temperature, current, showerhead height, and growth time)8,9,24,25. To produce carbon nanofibers longer than 25 &#181;m used by Morgan et al.7 and Davern et al.6, we increased the amount of Ni catalyst, altered the acetylene : ammonia ratio, and increased the current and the growth time. Additionally, we paid more attention to the geometry of the catalyst material. To produce tall straight fibers, the deposited catalyst needed to have a hockey puck shape rather than a shape that resembles a volcano (Figure 4). Volcano structures arise from remnants of photoresist after lift-off. To prevent the formation of volcanoes, a double layer of PMMA was used to create an undercut during electron beam lithography26. The undercut aids in lift-off of the deposited metal catalyst (Figure 2). The thick layer of the catalyst is important for the growth of tall VACNFs. The morphology of the VACNFs has been examined by Merkulova et al.24. The vertical alignment of VACNFs is due to both the Ni catalyst tip-type growth and the alignments of the DC potential perpendicular to the substrate (Figure 6). The showerhead describes the geometry of PECVD reactor (Figure 6) and serves as the source for the potential for the electric field27.
To define the array of catalyst dots with electron beam lithography, we applied an electron beam resist (polymethyl methacrylate), then used the e-beam to make small holes in the resist with a specific shape and in specific locations on the wafer. Holes of the desired diameter were placed on a regular grid with the defined spacing (pitch) and a file specifying the desired pattern was loaded into the electron beam lithography tool prior to loading the substrate into the machine. In addition to fiber height, another critical parameter for successful fiber transfer is the amount of time spent in the acetone bath. The VACNF films need to be left in the acetone bath long enough that their edges begin to curl; if they are left in the acetone bath for too little time, they are more difficult to lift off the chips and may break. The older the chips are, the longer they will have to remain in the acetone bath. Following the acetone bath, the films/chips were placed in isopropanol and water to remove access acetone as well as to remove the protective photoresist on the fibers.
To perform spin coating, wafers or wafer pieces are placed on a vacuum chuck in the spin coater, and the central position of the wafer is verified using the test function of the spin coater. A small puddle (~2.5 cm in diameter) of resist is applied to the center of the wafer and spun (3000 rpm for 45 s) Images of the fibers before and after spin coating are included in Figure 8 showing the preservation of fiber geometry (height, orientation, and pitch). The presence of fibers causes resist to well-up at the base of the fibers and results in thicker-than-expected layers. Spin-coating after VACNF growth has been explored by other groups11,18.
Another step within the process that is of vital importance is to ensure that the right amount of force is applied to VACNF chips/films. The delivery mechanism is dependent upon fibers making small punctures in cell walls via the impulse force of the tweezer tapping on rigid substrates6,7 or rolling with the mini-makeup applicator on flexible substrates. Fibers may or may not break off and remain imbedded in plant cells6,7without impact on the outcome, but practice in conjunction with examination for dye uptake and tissue damage is necessary to get the pressure right. Additionally, it is important to choose appropriate imaging time points after DNA delivery with VACNF chips/films as the time to detectable expression varies between plant species and the types of vectors being delivered7 (Figure 16).
As broadly applicable as this method is to plants, it has a few limitations. For example, adding a thin layer of silicon oxide to the VACNF films does not always result in films being completely hydrophilic because of the protective layer of photoresist added on top of the SU-8. If this problem materializes, thicker layers of silicon oxide could be applied to VACNFs. To test if the films are hydrophobic or hydrophilic, they can be placed in water. If the films sink, they are hydrophilic, and if they float, they are hydrophobic. Additionally, there can be variation between batches of fibers produced. There are several parameters that can be altered when growing the fibers in the dc -PECVD machine; what is described in this protocol is a set of parameters for two different amounts of Ni catalyst. Additionally, the crystal orientation of the Ni catalyst cannot be controlled28 and some branching will inevitably result in the fibers.
While we demonstrated the delivery of fluorescein dye and DNA to plant cells using both rigid and flexible substrates for this paper, the method should be broadly applicable for other biomolecules and genetic modification approaches, for example, RNAi silencing for plant systems like apples or other fruits where it would take years to produce stable transgenic lines. Moreover, these fibers could also be used to deliver genetic editing materials or for stable transformations in plants.

Plant transformation (both transient and stable) has yet to become widely achievable in all plant tissues and species. The transient transformation of plants is a process by which genes encoded in plasmids are temporarily introduced into plants but are not stably incorporated into the genome. Traditional methods that use particle bombardment, Agrobacteria, electroporation, or polyethylene glycol treatment of protoplasts are slow or can be cumbersome. Furthermore, they are not applicable to every plant species1,2,3,4.&#160;The use of nanomaterials for DNA delivery is a burgeoning field that is still in its infancy5. Nanomaterials, specifically carbon nanofibers have also successfully been used to deliver proteins, dextrans and dyes to plant leaves without causing wound response6. The goal of this work is to provide a detailed protocol for using one type of nanomaterial, carbon nanofibers, for delivering biomolecules or dyes to plants. Here, we focus on DNA as the biomolecule of choice, which permits the transient transformation of cells in various plant organs.
Previously, Morgan et al.7 demonstrated the use of carbon nanofibers affixed to rigid silicon substrate to transiently transform leaves of lettuce, N. benthamiana, and poplar, and both leaves and roots of Arabidopsis. Although transformations were successful on a variety of organs, fibers were more difficult to apply to plant tissues with curved surfaces, such as roots or fruits. We reasoned that a flexible backing for nanofibers might improve their efficiency of delivery by conforming better to the shape of the organ.
Herein, we detail methods utilized for fabricating and designing vertically aligned carbon nanofibers, transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants to deliver biomolecules and dyes. Carbon nanofibers were produced using direct current catalytic plasma-enhanced chemical vapor deposition (dc C-PECVD) with Ni catalyst. The position, diameter, and height of Ni catalyst dots were controlled using a combination of electron beam lithography, metal evaporation, and lift-off processes as described by Melechko et al.8,9. Using a double-layer e-beam resist, a thicker Ni catalyst can be deposited on the substrate to give longer fibers10. Fiber transfer from a rigid to a flexible substrate is based on a modification of methods described in Fletcher et al.11, with the current methods foregoing the use of an amorphous carbon layer or a sacrificial photoresist layer. SU-8 lift-off with fiber transfer is achieved by utilizing the intrinsic tensile stress resulting from underbaking and underexposing the SU-812,13,14. SU-8, a complex polymer, is naturally hydrophobic, which makes its use for facilitating DNA delivery difficult. To counteract the hydrophobic nature of SU-8, we apply a thin layer of silicon oxide via atomic layer deposition15 after fibers are embedded in SU-8. Application of fibers on a rigid substrate for biomolecule/dye delivery utilizes the impact force of tweezer tapping described in Davern et al.6 and the on-plant and on-chip methods described in Morgan et al.7. Flexible VACNF films are applied to curved plant surfaces by first semidrying DNA or dye droplets on the film as with the on-chip method from Morgan et al.7and then rolling the films on curved plant surfaces using a small makeup applicator16,17. Figure 1 depicts various approaches for applying fibers in rigid and flexible substrates to plants.

<table><tbody><tr><td>13&quot; x 13&quot; White 1/4-fold heavy duty Brawny industrial shop towel 70Ct</td><td>Fastenal&amp;nbsp;</td><td>690535</td><td></td></tr><tr><td>2-Propanol (IPA)&amp;nbsp;</td><td>Fischer Scientific</td><td>A451-4</td><td></td></tr><tr><td>4&quot; Lid</td><td>Entegris</td><td>H22-401-0615</td><td>Wafer Carriers</td></tr><tr><td>4&quot; tray</td><td>Entegris</td><td>H22-40-0615</td><td>Wafer Carriers</td></tr><tr><td>Accretech SS10 dicing saw</td><td>Accreteck</td><td>SS10</td><td></td></tr><tr><td>Acetone</td><td>Fischer Scientific</td><td>A18-4</td><td></td></tr><tr><td>Acetone used in the cleanroom at ORNL</td><td>JT Baker</td><td>9005-05</td><td></td></tr><tr><td>Apples</td><td>Grocery store</td><td>No product number</td><td></td></tr><tr><td>Arabidopsis thaliana&amp;nbsp;</td><td>Seeds of accession Columbia from the laboratory of Professor Jean Greenberg at the University of Chicago</td><td>No product number</td><td></td></tr><tr><td>Carbon direct current plasma enhanced chemical vapor deposition machine</td><td>Oak Ridge National Laboratory&amp;nbsp;</td><td>Custom-built</td><td></td></tr><tr><td>Cobham Green lettuce&amp;nbsp;</td><td>Seeds from the laboratory of Professor Richard Michelmore at the University of California, Davis</td><td>No product number</td><td>Butterhead lettuce</td></tr><tr><td>Fluorescein dye</td><td>Sigma Aldrich</td><td>F2456-2.5G</td><td></td></tr><tr><td>Gel-box&amp;nbsp;</td><td>Gel-Pak</td><td>&amp;nbsp;AD-23C-00-X4</td><td></td></tr><tr><td>Heidelberg DWL 66 direct-write lithography tool&amp;nbsp;</td><td>Heidelberg</td><td>DWL 66</td><td></td></tr><tr><td>ImageJ</td><td>National Institues of Health&amp;nbsp;</td><td>No product number</td><td></td></tr><tr><td>Isoproponal (IPA) used in the cleanroom at ORNL</td><td>Doe and Ingalls</td><td>CMOS Grade 9079-05</td><td></td></tr><tr><td>JEOL 9300FS 100kV electron beam lithography system</td><td>JEOL</td><td>8100</td><td></td></tr><tr><td>Kimwipes</td><td>Kimtech</td><td>Kimberly-Clark Professional 34120</td><td></td></tr><tr><td>Kord-Valmark disposable polystyrene petri dish</td><td>VWR</td><td>11019-554</td><td></td></tr><tr><td>Layout Editor&amp;nbsp;</td><td>&amp;nbsp;juspertor GmbH</td><td>No product number&amp;nbsp;</td><td></td></tr><tr><td>LSM 710 confocal microscope</td><td>Zeiss</td><td>No product number</td><td></td></tr><tr><td>LSM 800 confocal microscope</td><td>Zeiss</td><td>No product number</td><td></td></tr><tr><td>Make-up applicator&amp;nbsp;</td><td>Amazon</td><td>G2PLUS</td><td>500 PCS Disposable Micro Applicators Brush for Makeup and Personal Care (Head Diameter: 1.5 mm)- 5 x 100 PCS</td></tr><tr><td>Merlin field emission scanning electron microscope</td><td>Zeiss</td><td>Merlin</td><td></td></tr><tr><td>MIBK/IPA&amp;nbsp; (methyl isobutyl ketone/isopropanol) (1:3)</td><td>Microchem</td><td>M089025</td><td></td></tr><tr><td>Onions</td><td>Grocery store</td><td>No product number</td><td></td></tr><tr><td>Oxford FlexAl atomic layer deposition</td><td>Oxford</td><td>FlexAl</td><td></td></tr><tr><td>PMMA 495 A4</td><td>Microchem</td><td>M130004</td><td></td></tr><tr><td>PMMA 950 A4</td><td>Microchem</td><td>M230004</td><td>Can dilute down to A2</td></tr><tr><td>Polyethylene terephthalate (PET)</td><td>Amazon</td><td>KS-6304-21-11</td><td>Type D Clear PET (Polyester) Sheet .0005&quot; Thick x 27&quot; Width x 10 Ft Length 1 pc</td></tr><tr><td>Precision tweezers</td><td>Aven Inc.&amp;nbsp;</td><td>18032TT</td><td></td></tr><tr><td>pUBQ10:YFP-GW</td><td>Arabidopsis Biological Resource Center</td><td>CD3-1948</td><td></td></tr><tr><td>Silicon etcher (used for descum)&amp;nbsp;</td><td>Oxford</td><td>Plasmalab</td><td></td></tr><tr><td>Silicon rubber kit</td><td>Smooth-On Inc</td><td>Ecoflex 00-20</td><td></td></tr><tr><td>Silicon wafers</td><td>Pure Wafer</td><td>4N0.001-.005SSP-INV</td><td></td></tr><tr><td>Spin coater</td><td>Brewer Sciences</td><td>Model 100CB</td><td></td></tr><tr><td>SPR 955cm 0.7&amp;nbsp;</td><td>Megaposit</td><td>10018314</td><td></td></tr><tr><td>Strawberries</td><td>Grocery store</td><td>No product number</td><td></td></tr><tr><td>SU-8 2015</td><td>Microchem</td><td>SU-8 2000 Series</td><td>Toxic. Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td></tr><tr><td>SU-8 developer</td><td>Microchem</td><td>SU-8 2000 Series</td><td>Handle with care. Wear chemical goggles, chemical gloves and suitable protective clothing when handling SU-8 2000 resists. Do not get into eyes, or onto skin or clothing.</td></tr><tr><td>Suss MicroTec contact aligner</td><td>Suss MicroTec</td><td>MA6/BA6</td><td></td></tr><tr><td>Table top microscope</td><td>Phenom XL</td><td>used for checking Ni catalysts after metal deposition</td><td></td></tr><tr><td>Thermionics VE-240 e-beam evaporator</td><td>Thermionics</td><td>VE-240</td><td></td></tr></tbody></table>

using vertically aligned carbon nanofiber arrays on rigid or flexible substrates for delivery of biomolecules and dyes to plants

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 &#181;L droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Watch this Scientific Journal Video about Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays at JoVE.com

Author Spotlight: Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays 

Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery.

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to ...

using-vertically-aligned-carbon-nanofiber-arrays-on-rigid-or-flexible

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.4K Views.  The University of Chicago. We proberen een algemene methode te demonstreren voor het afleveren van kleurstof of DNA in planten met behulp van koolstofnanovezelarrays. Veel planten zijn recalcitrant ten opzichte van traditionele transformatiemethoden. Onze methode kan dit probleem oplossen met behulp van koolstofnanovezelarrays op flexibele, stijve substraten.Onze techniek is snel en gebruiksvriendelijk. Het kan worden toegepast op vrijwel elke plantensoort of plantenorgaan. Het kan ook worden gebruikt om andere ...

Video: Auteur in de schijnwerpers: Fabriekstransformatie ontgrendelen door te innoveren met koolstofnanovezelarrays 

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Genetics

Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates

In recent years &pi;-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.	
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently &pi;-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important &pi;-conjugated small molecular weight organics, such as tris-8-hydroxyquinoline aluminum (Alq3), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.	
Recently we have been able to address the above-mentioned issue by employing a novel "centrifugation-assisted" approach. This method therefore broadens the spectrum of organic materials that can be patterned in a vertically ordered nanowire array. Due to the technological importance of Alq3, rubrene and methanofullerenes, our method can be used to explore how the nanostructuring of these materials affects the performance of aforementioned organic devices. The purpose of this article is to describe the technical details of the above-mentioned protocol, demonstrate how this process can be extended to grow small-molecular organic nanowires on arbitrary substrates and finally, to discuss the critical steps, limitations, possible modifications, trouble-shooting and future applications.

We report a simple method for fabricating an ultrahigh density array of vertically ordered small-molecular organic nanowires. This method allows for synthesis of complex heterostructured hybrid nanowire geometries, which can be inexpensively grown on arbitrary substrates. These structures have potential applications in organic electronics, optoelectronics, chemical sensing, photovoltaics and spintronics.

In  recent years &pi;-conjugated  organic semiconductors have emerged as the active material in a number of  diverse applications including large-area, ...

Engineering

Fabrication of Low Temperature Carbon Nanotube Vertical Interconnects Compatible with Semiconductor Technology

We demonstrate a method for the low temperature growth (350 &#176;C) of vertically-aligned carbon nanotubes (CNT) bundles on electrically conductive thin-films. Due to the low growth temperature, the process allows integration with modern low-&#954; dielectrics and some flexible substrates. The process is compatible with standard semiconductor fabrication, and a method for the fabrication of electrical 4-point probe test structures for vertical interconnect test structures is presented. Using scanning electron microscopy the morphology of the CNT bundles is investigated, which demonstrates vertical alignment of the CNT and can be used to tune the CNT growth time. With Raman spectroscopy the crystallinity of the CNT is investigated. It was found that the CNT have many defects, due to the low growth temperature. The electrical current-voltage measurements of the test vertical interconnects displays a linear response, indicating good ohmic contact was achieved between the CNT bundle and the top and bottom metal electrodes. The obtained resistivities of the CNT bundle are among the average values in the literature, while a record-low CNT growth temperature was used.

A method for the growth of low temperature vertically-aligned carbon nanotubes, and the subsequent fabrication of vertical interconnect electrical test structures using semiconductor fabrication is presented.

We demonstrate a method for the low temperature growth (350 &#176;C) of vertically-aligned carbon nanotubes (CNT) bundles on electrically conductive ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Well-aligned Vertically Oriented ZnO Nanorod Arrays and their Application in Inverted Small Molecule Solar Cells

This manuscript describes how to design and fabricate efficient inverted solar cells, which are based on a two-dimensional conjugated small molecule (SMPV1) and [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM), by utilizing ZnO nanorods (NRs) grown on a high quality Al-doped ZnO (AZO) seed layer. The inverted SMPV1:PC71BM solar cells with ZnO NRs that grew on both a sputtered and sol-gel processed AZO seed layer are fabricated. Compared with the AZO thin film prepared by the sol-gel method, the sputtered AZO thin film exhibits better crystallization and lower surface roughness, according to X-ray diffraction (XRD) and atomic force microscope (AFM) measurements. The orientation of the ZnO NRs grown on a sputtered AZO seed layer shows better vertical alignment, which is beneficial for the deposition of the subsequent active layer, forming better surface morphologies. Generally, the surface morphology of the active layer mainly dominates the fill factor (FF) of the devices. Consequently, the well-aligned ZnO NRs can be used to improve the carrier collection of the active layer and to increase the FF of the solar cells. Moreover, as an anti-reflection structure, it can also be utilized to enhance the light harvesting of the absorption layer, with the power conversion efficiency (PCE) of solar cells reaching 6.01%, higher than the sol-gel based solar cells with an efficiency of 4.74%.

This manuscript describes how to design and fabricate efficient inverted SMPV1:PC71BM solar cells with ZnO nanorods (NRs) grown on a high quality Al-doped ZnO (AZO) seed layer. The well-aligned vertically oriented ZnO NRs exhibit high crystalline properties. The power conversion efficiency of solar cells can reach 6.01%.

This manuscript describes how to design and fabricate efficient inverted solar cells, which are based on a two-dimensional conjugated small molecule ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

Environment

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Molecule Delivery in Plants Using a Direct Infusion Device

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

Author Spotlight: Direct Infusion Device for Molecule Delivery in Plants  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

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