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

The distinct advantage of VACNF chips on rigid or flexible substrates is the ability to deliver biomolecules or dyes to specific locations on a plant (Figure 1). Here, we used fluorescence readouts to assess delivery. Using different plants, substrates, and delivery methods (on-chip or on-plant), there can be differences in the timing of the appearance of dye fluorescein. To determine whether VACNF chips/films work for delivery, a fast approach is to use fibers for dye delivery (Figure 9). Images labeled with different times in Figure 9 are different fields of view from the same samples. When using on-plant method, fluorescein dye can be observed immediately after delivery using fluorescence microscopy. Additionally, if the same field of view is imaged over time after delivering fluorescein dye to a plant, the signal intensity becomes less bright over time (Figure 10). The fluorescein dye could potentially be moving from the field of view to other areas of the leaf through plasmodesmata^20,21. Compared to the on-plant method, in the on-chip method with fibers on the rigid substrate, the dye moves slower throughout the impaled area (Figure 9). This could be a result of the dye detaching from fibers/rehydrating in the cells and thus taking more time to move.

Fibers in the flexible substrate were suitable for dye delivery to curved surfaces such as strawberries and apples (Figure 11 and Figure 12). Using a flexible substrate with strawberries, a strong fluorescein signal was observed right away (Figure 11), whereas it took 2 h to see a strong fluorescein signal in apples (Figure 12B,D). The successful delivery of plasmids encoding fluorescent markers can be determined using fluorescence microscopy to find the resulting signal (Figure 12F, Figure 13, and Figure 14). Controls are necessary to determine whether the plant of choice has no autofluorescence similar to the fluorescent marker we want to deliver by fibers. Using fibers alone is helpful to determine if the impact force applied with tweezers results in damage to the plant tissue or if the observed fluorescence within the sample is due to the VACNFs, which are inherently fluorescent because of their silicon nitride layer¹⁸ (Figure 13C–D). Fibers impaled in plant tissue do not induce wound response as assessed by H₂O₂ production⁶. Lastly, using the control of +DNA, -Fibers is necessary to confirm that DNA is not entering into the plant through tapping alone and confirms that the fibers are necessary for delivery into plant cells (Figure 13E–F). There should be no distinct difference when using the on-plant or on-chip methods of delivery using the VACNFs on a rigid substrate, as indicated by the lack of significant difference in fluorescence values (Figure 13I). Using the flexible VACNF films with on-chip DNA delivery resulted in a successful transient transformation of epidermal cells in store-bought apples and onions (Figure 12F and Figure 14).

Failed experiments may have fibers broken off in different fields of view, but there will be no resulting fluorescence signal from the attempt to deliver plasmid DNA or dye. If too much pressure is applied to the plant, there will be apparent tissue damage (Figure 15). This mechanical damage may look like small holes or transparent areas in the plant as if a layer of cells has been removed when looking at the plant under a microscope. Sometimes imprints of the chip will be visible. An experiment in which fluorescent protein expression is not detected after DNA delivery may also be due to the use of low-quality DNA, so it may be useful to prepare fresh DNA.

Figure 1: Schematic of dye/DNA delivery to plant tissue using fibers on rigid and flexible substrates. (A) On−plant, fiber-mediated dye/DNA delivery. A 1-µL droplet of dye/DNA solution is placed on the surface of a leaf and the VACNF chip is placed on top of the droplet. Using a pair of tweezers, the chip is gently tapped into the tissue. The rigid substrate is removed leaving nanofibers within the tissue. (B) On-chip fiber-mediated DNA delivery. A 1 µL droplet of dye or DNA solution is drop cast on the VACNF chip and dried for 15 min. The chip with semidried DNA is placed on top of the leaf surface and tapped into the tissue as in panel A. (C) On-chip SU-8 film DNA delivery. Fibers are transferred from the rigid silicon substrate to flexible SU-8 backing. A 1 µL droplet of dye/DNA solution is drop cast on the VACNF film and dried for 10 min. The VACNF film with the semidried dye/DNA is then rolled onto a curved plant surface using a makeup applicator. Please click here to view a larger version of this figure.

Figure 2: Workflow for making vertically aligned carbon nanofiber arrays. To produce VACNFs, a double layer of polymethyl methacrylate (PMMA 495 A4 followed by PMMA 950 A2) is spin-coated onto a silicon wafer. Electron beam lithography is used to define an array of dots 300 nm in diameter. The resist is then developed in methyl isobutyl ketone/isopropanol (MIBK/IPA), 1:3 for 1.5 min. Using a metal evaporator, an adhesive layer of Ti (10 nm) is applied to the wafer, followed by a layer of Ni (130 nm or 150 nm). The remaining resist is then removed via lift-off (bath sonication in acetone). The geometry of catalyst dots is inspected via SEM. If the catalysts resemble hockey pucks and are flat, they are placed in the plasma enhanced chemical vapor deposition (PECVD) machine and fibers are grown. The fibers were then inspected using SEM. Please click here to view a larger version of this figure.

Figure 3: SEM images of ideal vertically aligned carbon nanofibers. (A) Electron micrograph of VACNFs with a ~35 µm pitch and a height of 10-15 µm, imaged at a 30° angle. This image is duplicated in Figure 7C. (B) Electron micrograph of VANCFs with ~20 µm pitch and 20-30 µm height, and imaged at a 30° angle. (C) Electron micrograph of VANCFs with ~35 µm pitch, 50-60 µm height, and imaged at a 30° angle. (D) Electron micrograph of VACNF tip with a diameter <200 nm. There is variation in the fiber tip diameters (150-300 nm). Because the fibers were imaged at 30° angle, the heights appear to be smaller than the actual height by a factor of sin(30°)=1/2. Panels A and B have been reprinted with permission from Morgan et al.⁷. Please click here to view a larger version of this figure.

Figure 4: SEM images of catalyst geometry prior to growing VACNFs. (A) SEM image of the catalyst after lift-off. Note that photoresist is present around the rim of the catalyst, resulting in a volcano shape. (B,C) SEM images show the volcano shapes of the catalyst after using a single-layer PMMA resist. (D) Desired hockey puck catalyst shape made from double layers of PMMA. Because the fibers were imaged at 30° angle in panels A, B, and D, the heights appear to be smaller than the actual height by a factor of sin(30°) = 1/2. Scale bars represent 100 nm. Please click here to view a larger version of this figure.

Figure 5: Effect of catalyst dot size on fiber growth: To establish the optimal diameter of the catalyst material, fibers were grown from dot sizes ranging from 200-600 nm. Dot sizes ranging from 400-600 nm led to dewetting of the catalyst and the growth of multiple fibers. The best fiber geometry was produced by 300 nm diameter. Smaller dots led to insufficient fiber height. Due to the fact that the fibers were imaged at 30° angle, the heights appear to be smaller than the actual height by a factor of sin(30°) = 1/2. Images were taken using scanning electron micrograph. Please click here to view a larger version of this figure.

Figure 6: Schematic illustration of the direct current – plasma enhanced chemical vapor deposition (dc-PECVD) system used at Oak Ridge National Laboratory (ORNL). The custom system at ORNL has a large showerhead that serves as the reactor for feed gases and output for plasma. The showerhead was maintained at a DC potential of 300 V relative to the heated stage for the substrate. Please click here to view a larger version of this figure.

Figure 7: Workflow for transferring fibers from a rigid substrate to a flexible substrate. After nanofiber synthesis and inspection, each wafer is spin-coated with SU-8 2015 at 4000 rpm for 45 s. The wafer is then soft-baked for 3 min at 95 °C. Then the wafer is exposed to UV light and patterned in a mask aligner at 95 mJ/cm². After postbaking for 6 min at 95 °C, the wafer is developed in the SU-8 developer for 15 s, rinsed with IPA, and dried with N₂ gas. A protective resist layer of SPR 955 CM 0.7 is spin-coated on the wafer at 3000 rpm and softbaked at 90 °C for 30 s. A silicon oxide layer (2-3 nm) is then added to the wafer via atomic layer deposition (ALD) (22 cycles at 100 °C) to make the flexible substrate hydrophilic¹⁵. The wafer is then diced into 3 mm x 3 mm squares with a dicing saw. At the time of use, individual chips are placed in acetone until SU-8 starts to curl and becomes concave (>30 min). At this time, the SU-8 layer on most chips can be grabbed at the edge with sharp tweezers and peeled from the underlying silicon substrate as an intact 3 mm square film. The film is then washed successively in IPA and water for 5 min each and used immediately. Please click here to view a larger version of this figure.

Figure 8: SEM images of fiber transfer from a rigid substrate to a flexible substrate. Images shown are representative but come from different samples. (A) Long fibers after PECVD growth (same image as in Figure 2C). (B,C) Fibers after application of SU-8. The epoxy wells up around the base of fibers. The exposed fiber length ranged from 5 µm to 30 µm. (D) Fibers embedded in Su-8 after lift-off retained their geometry. Due to the fact that the fibers were imaged at 30° angle, the heights appear to be smaller than the actual height by a factor of sin(30°)=1/2. Panel A has been reprinted with permission from Morgan et al.⁷. Please click here to view a larger version of this figure.

Figure 9: Dye delivery to Arabidopsis leaves using on-chip and on-plant methods with fibers on rigid substrates. (A-P) Images were acquired using confocal microscopy. On-chip method (A–H): 1 µL of 10 µM fluorescein dye was dried for 15 min on VACNF chips, and then the chips were tapped into the abaxial side of Arabidopsis leaves with tweezers. (A–D) Leaves imaged within 5-15 min post-delivery. (E–H) Leaves imaged 1 h after delivery. (A,E) +Dye, +Fibers. Controls: (B,F) -Dye, -Fibers; (C,G) -Dye, +Fibers; (D,H) +Dye, -Fibers. (I–P) On-plant method: 1 µL of 10 µM fluorescein dye was placed on the plant surface, chips were positioned such that they came into contact with the droplet, and tweezers were used to tap the chip into the abaxial side of Arabidopsis leaves. (I–L) imaged within 5-15 min post-delivery and (M–P) imaged 1 h after delivery. (I,M) +Dye, +Fibers. Controls: (J,N) -Dye, -Fibers; (K,O) -Dye, +Fibers; (L,P) +Dye, -Fibers. Panels A-P are single planar images from z-stacks. Scale bars are 20 µm. Fibers have a 35 µm pitch. Please click here to view a larger version of this figure.

Figure 10: Time course of dye delivery in Arabidopsis via on-plant method with rigid substrate. Images were acquired using confocal microscopy. Using the on-plant method, 1 µL droplet of fluorescein dye solution (10 µM) was placed on the surface of a leaf, and the VACNF chip was placed on top of the droplet. Using a pair of tweezers, the chip was gently tapped into the tissue. +Dye, +Fibers images of the same area were acquired every 5 min. Scale bars are 20 µm. Fibers have a 35 µm pitch. Panels are single planar images from z-stacks. Please click here to view a larger version of this figure.

Figure 11: Dye delivery to strawberry fruit using VACNF films. (A-H) Images were acquired using confocal microscopy. Using the on-chip method, dye droplets were dried on VACNF films, which were then rolled onto fruit surfaces using a makeup applicator. Fluorescein dye (10 µM) was delivered to strawberry cells and imaged after (A) 10 min and (B) 1 h. (C,D) No treatment controls (-Dye, -Fibers). (E,F) -Dye, +Fibers controls after 10 min and 1 h, respectively. (G,H) +Dye, -Fibers controls after 10 min and 1 h, respectively. The scale bars are 40 µm. Panels A-H are maximum projections of 188 µm z-stacks. Fibers have a 35 µm pitch. Please click here to view a larger version of this figure.

Figure 12: Dye delivery and transient transformation of apples via VACNF films. (A-F) Images were generated using confocal microscopy. Using the on-chip method, 1 µL droplets of fluorescein dye (10 µM, B and D) or 1 µL of plasmid encoding pUBQ₁₀:YFP (DNA−YFP) (200 ng) were dried on VACNF films, which were then rolled onto fruit surfaces using a makeup applicator. Fluorescein dye was delivered to the apple epidermis and imaged after (B) 10 min and (D) 2 h. (D) The dye took some time to diffuse into the cells after delivery. DNA-YFP delivery and expression via VACNF films after (F) 48 h. (A,C,E) no treatment controls (-Dye/DNA-YFP, -Fibers). The scale bars are 40 µm. Panels A-D are single planar images from z-stacks. Panels E and F are the maximum projection of 53 µm z-stacks. The fiber pitch is 35 µm. Arrows denote fluorescein or YFP signals. * indicates fibers. Please click here to view a larger version of this figure.

Figure 13: Transient transformation of Arabidopsis leaves using on−plant or on−chip VACNFs methods. (A–H) Images were acquired using confocal microscopy 48 h after DNA delivery. (A,C,E,G) On-plant method: 1 µL of plasmid encoding pUBQ₁₀:YFP (DNA−YFP) (200 ng) was placed on the abaxial side of Arabidopsis leaves. Chips were positioned such that they came in contact with the droplet, and tweezers ware used to tap the chip into leaf tissue. (B,D,F,H) On-chip method : 1 µL of DNA−YFP (200 ng) was dried for 15 min on VACNF chips and then the chips ware tapped into the abaxial side of Arabidopsis leaves with tweezers. +DNA-YFP, +Fibers for (A) on-plant and (B) on-chip. Controls: (C,D) -DNA-YFP, + Fibers; (E,F) +DNA-YFP, -Fibers; and (G,H) -DNA, −Fibers. (I) Graph of relative average fluorescence signal intensity of 25 20 × 20 µm areas from images of 5 biological replicates combined from 2-3 experiments using fluorescence from the YFP channel. Regions containing stomata (*) were excluded due to autofluorescence. The average fluorescence intensity from the -DNA-YFP, -Fibers condition was subtracted from each average. 2−way ANOVA (and Tukey test) was used for significance testing, and error bars represent the standard error of the mean. Different letters show significant differences between treatments (P < 0.0001). All images shown are maximum projections of 40 µm z−stacks. Scale bars are 20 µm. White arrows indicate fibers in the images. The fiber pitch is 35 µm. This figure has been reprinted with permission from Morgan et al.⁷. Please click here to view a larger version of this figure.

Figure 14: Transient transformation of onions using VACNF films. Images were acquired using confocal microscopy 48 h after DNA delivery. Using the on-chip method, 1 µL plasmid DNA encoding pUBQ₁₀:YFP (DNA-YFP) (200 ng) droplets were dried on VACNF films for 10 min, which were then rolled onto plant organ surfaces. (A) DNA-YFP was delivered to, and YFP was expressed in the onion epidermis. (B) No treatment control; (C) control (+DNA-YFP, -Fibers) and (D) control (-DNA-YFP, +Fibers). The scale bars are 40 µm. Fibers have a 35 µm pitch. Images are maximum projections of 115 µm z-stacks. Please click here to view a larger version of this figure.

Figure 15: Tissue damage in lettuce from VACNF film application. (A-D) Images were generated using confocal microscopy. (A) The on-chip method was used to deliver DNA to lettuce leaves via VACNF films. pUBQ₁₀:YFP DNA (200 ng) droplets were dried for 10 min on VACNF films, which were then rolled onto the abaxial side of detached lettuce leaves and stored in a humidity chamber for 4 days. (B) control (-DNA-YFP, +Fibers). (C) control (+DNA-YFP, -Fibers), and (D) no treatment (-DNA-YFP, -Fibers). Scale bars are 40 µm. VACNFs have a 35 µm pitch. Arrows point to plant damage resulting from rolling of the flexible substrate with too much force. Note that successful VACNF-mediated DNA delivery in lettuce was achieved in other experiments⁷. Please click here to view a larger version of this figure.

Figure 16: Workflow of VACNF-mediated delivery in plants. In Stage I, check the geometry of the fibers using SEM. For proper delivery, fibers need a tip with a diameter <200 nm. If using fibers on flexible substrate, the next step would be to confirm that fibers are being transferred to SU-8 and to check the height of the exposed fibers via SEM. In Stage II, we tested the usefulness of the fibers by trying to deliver dye into the plant/organ of choice using a rigid or flexible substrate. Use 1 µL droplet, either placing it on the plant surface or briefly drying it on the chip/film. With this step and all other steps, it is imperative to use the proper controls (-Dye,-Fibers; -Dye, +Fibers; and +Dye,-Fibers) to be confident that the signal comes from bona fide dye delivery. In Stage III, confirm that the gene of interest is present in the plasmid, determine the concentration of DNA to deliver, and test the optimal amount of time after delivery to check expression. In Stage IV, the outcome was evaluated using confocal microscopy to check the expression of the delivered marker. This figure has been modified with permission from Morgan et al.⁷. Please click here to view a larger version of this figure.