Arrays of silicon lances were made using photolithographic and STS DRIE Bosch techniques. Arrays consist of a 10 mm square grid pattern of lances measuring 100 m tall and having a 3 mm diameter, each lance being spaced 30 mm apart. The tips of lances are pointed, allowing easier penetration through plant cell walls. A nanoinjector device was also made to accept the silicon lance arrays and perform nanoinjections. A nanoinjection consisted of 2 silicon lance arrays, with lances oriented towards each other, being moved into and out of a plant cotyledon placed between them. Prior to the nanoinjection, polar molecules in solution can be attracted to the lances through a process utilizing the nanoinjector device’s ability to control the electrical current between the 2 lance arrays. During the nanoinjection the displacement between the lances, the force exerted on the plant cotyledon and the electrical current between the lance arrays are controlled. Once the lances are inserted into the cells, the electrical current between the lance arrays is reversed, repelling the molecular load from the lance array. Propidium iodide (PI) and Cotton Leaf Crumple Virus (CLCrV) were used as molecular loads in nanoinjections. The nanoinjector also records and outputs data from the nanoinjection for analysis. Nanoinjections were performed on Arabidopsis and Cotton cotyledons. Changes in the force applied during a nanoinjection and varying the number of repeated nanoinjections on the same cotyledon were observed. Too much force or too many repeated injections causes physical damage to the cotyledon. An optimal force and number of repeated injections can be performed without causing physical damage to the cotyledon. Successful transfection of PI and CLCrV was not observed in a relatively small number of performed nanoinjection procedures on either Arabidopsis or Cotton cotyledons. Possible interacting variables and recommendations for further work are discussed.
BYU ScholarsArchive Citation
Brown, Taylor Andrew, "In Vivo Silicon Lance Array Transfection of Plant Cells" (2020). Theses and Dissertations. 8398.
nanoinjection, transfection, CRISPR, silicon, injection, lance, array