Nanostraw platform for nondestructive intracellular sampling and precise delivery
Abstract/Contents
- Abstract
- Nanotechnology has shown great potential in biological, medical and clinical applications. In this thesis, I will describe two novel bio-applications based on a recently-developed nanostraw platform. In the first part of the thesis, I will describe a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 days through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with > 95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual content within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activities such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to have a sight into the dynamic cell behavior, including processes such as induced pluripotency and differentiation. In the second part, I will elaborate a controlled quantitative intracellular delivery by electroporation-assisted alumina NS system (NES). The cell populations that form intimate contact with NS experience localized electric field during delivery and are porated on the tip of the NS. We achieved a quantitative and high uniform delivery of mRNAs and proteins by controlling three variables: (1) delivery duration, (2) electric pulse intensity, and (3) delivery reagent concentration. Fast protein transcription was observed soon after delivery. We also demonstrated ratiometric co-transfection of mRNAs, and showed this system allows high rate transfection to multiple cell lines and difficult-to-transfect primary cells. The NES transfection efficiency is independent of cell density. In addition, we monitored protein delivery and subsequent biological activity, including STIM1 and antibodies. We observed STIM1 peptide binding to cell expressed ORAI protein, immune antibody staining on histone and actin filaments.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Cao, Yuhong |
|---|---|
| Associated with | Stanford University, Department of Materials Science and Engineering. |
| Primary advisor | Melosh, Nicholas A |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Melosh, Nicholas A |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Cui, Yi, 1976- |
| Advisor | Cui, Yi, 1976- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Yuhong Cao. |
|---|---|
| Note | Submitted to the Department of Materials Science and Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Yuhong Cao
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...