on-plant method to deliver dye or biomolecules using vacnfs in a rigid substrate

Uso de matrices de nanofibras de carbono para la entrega eficiente de biomoléculas y colorantes a las plantas

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.

Autor destacado: Desbloqueando la transformación de las plantas mediante la innovación con matrices de nanofibras de carbono 

Uso de matrices de nanofibras de carbono alineadas verticalmente sobre sustratos rígidos o flexibles para la entrega de biomoléculas y colorantes a las plantas

We are trying to demonstrate a general method for delivery of dye or DNA into plants using carbon nanofiber arrays. Many plants are recalcitrant to traditional transformation methods. Our method can solve this problem using carbon nanofiber arrays on flexible rigid substrates.Our technique is fast and user-friendly. It can be applied to virtually any plant species or plant organ. It can also be used to deliver other reagents such as dyes and proteins.

Watch this Scientific Journal Video about On-Plant Method to Deliver Dye or Biomolecules Using VACNFs in a Rigid Substrate at JoVE.com

On-Plant Method to Deliver Dye or Biomolecules Using VACNFs in a Rigid Substrate  

Método en planta para administrar colorantes o biomoléculas utilizando VACNF en un sustrato rígido

Para comenzar, retire la fotoresistencia de las VACNF lavándolas con acetona al 100 % durante cinco minutos, seguidas de un lavado de cinco minutos con IPA al 100 % y luego agua destilada dos veces durante cinco minutos. Luego, coloque el tejido vegetal que se va a empalar sobre una superficie dura como soporte. Coloque una gota de tinte de un microlitro sobre la superficie del tejido vegetal.Coloque un chip VACNF con un sustrato rígido en la parte superior de la gota, con las fibras orientadas de tal manera que entren en contacto con la gota. Usando el lado plano de un par de pinzas, golpee el chip, marque el área de la planta con la que el chip entró en contacto con un marcador de punta suave. Retire los chips VACNF después de la entrega.Repita estos pasos para los controles, como colocar el tinte en el lado sin fibra de una película, usar la película sin tinte y usar el lado sin fibra de una película. Almacene las plantas intactas o los órganos de plantas extirpados en cámaras húmedas en condiciones de días prolongados. Para los órganos extirpados, use una placa de Petri de plástico con toallas de papel húmedas.Usando este método, se administró colorante de fluoresceína a las hojas de Arabidopsis. Las imágenes tomadas en diferentes momentos mostraron que el tinte de fluoresceína se observó inmediatamente después del parto. De manera similar, se logró la liberación de un plásmido que contenía proteína fluorescente amarilla en hojas de Arabidopsis.

on-plant-method-to-deliver-dye-or-biomolecules-using-vacnfs-rigid

Research

JoVE Journal

Bioengineering

On-Plant Method to Deliver Dye or Biomolecules Using VACNFs in a Rigid Substrate

233 vistas.  The University of Chicago. Para comenzar, retire la fotoresistencia de las VACNF lavándolas con acetona al 100 % durante cinco minutos, seguidas de un lavado de cinco minutos con IPA al 100 % y luego agua destilada dos veces durante cinco minutos. Luego, coloque el tejido vegetal que se va a empalar sobre una superficie dura como soporte. Coloque una gota de tinte de un microlitro sobre la superficie del tejido vegetal.Coloque un chip VACNF con un sustrato rígido en la parte superior de la gota, con las fibras...

Video: On-Plant Method to Deliver Dye or Biomolecules Using VACNFs in a Rigid Substrate  

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.

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.

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 ...

Investigación

Bioingeniería

On-Chip Method to Deliver Dye or Biomolecules to Plants Using VACNFs in a Flexible Substrate

Begin by preparing vertically aligned carbon nano fibers, or VACNFs. To transfer the VACNFs to a flexible substrate, first place the individual chips in acetone to separate the flexible substrate from the rigid substrate. Then, wash SU-8 films with IPA for five minutes, followed by washing with water for five minutes.Place the chips in a commercial box with a sticky pad when transporting them. Prepare a silicone rubber PET holder by mixing the elaststomer and the crosslinker from a two-part kit. Cut out a square of PET and tape it down in a clear plastic dish.Then, pour a very thin film of silicone rubber on top of the PET and cure it at 80 degrees Celsius for one to two hours. Transfer the SU-8 film to the PET holder using a pair of tweezers, and place the fiberless side of the SU-8 film on the silicone rubber. Press on the edges of the SU-8 square to help it stick to the silicone rubber PET and avoid breaking off fibers.Then, place a one microliter droplet of dye onto the fiber side of the SU-8 film and let it dry for 10 minutes. After drying, place the VACNF film onto the plant surface using a pair of sharp tweezers. Then, gently roll a small makeup applicator over the VACNF film.Mark the areas where the flexible substrates are placed with a soft tipped marker. Then remove the flexible substrates from the plant surface using tape. Repeat VACNF application for controls, such as placing dye on the fiberless side of a film, using the film without dye, and using the fiberless side of a film.The on-chip method was used to deliver fluorescein dye to curved surfaces, such as strawberries, in which a strong fluorescein signal was observed immediately. Whereas in apples, a strong fluorescein signal was seen after two hours of delivery. Similarly, delivery of a plasmid containing yellow fluorescent protein was achieved in apples.Also, a successful transient transformation of onions was done by delivering the plasmid containing yellow fluorescent protein using VACNF films.

Método en chip para administrar colorantes o biomoléculas a las plantas utilizando VACNF en un sustrato flexible

To begin, remove the photo resist from the VACNFs by washing with 100%acetone for five minutes, followed by a five minute wash with 100%IPA, and then double distilled water for five minutes. Then, place the plant tissue to be impaled on a hard surface for support. Place a one microliter droplet of dye onto the surface of the plant tissue.Place a VACNF chip with a rigid substrate down the top of the droplet, with the fibers oriented such that they will come into contact with the droplet. 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.Repeat these steps for controls, such as placing dye on the fiberless side of a film, using the film without dye, and using the fiberless side of a film. Store intact plants or excised plant organs in humid chambers in long day conditions. For excised organs, use a plastic Petri dish with wet paper towels.Using this method, fluorescein dye was delivered to Arabidopsis leaves. Images taken at different time points, showed that the fluorescein dye was observed immediately after delivery. Similarly, delivery of a plasmid containing yellow fluorescent protein was achieved in Arabidopsis leaves.