Abstract
Structures on the nanoscale, such as quantum dots, carbon nanotubes, or nanowires, are highlyinteresting to apply in biological research, since their nanoscale dimension is compatible withcrucial biomolecules, such as proteins, and several orders of magnitude smaller than amammalian cell. Among these, arrays of vertical nanostructures (NSs) with submicron diametersand microscale lengths are particularly promising and are currently being established as bothhighly sensitive protein arrays and as platforms for manipulations and investigations at thesingle-cell or even subcellular level.To date, a wealth of NS array materials, geometries and cellular applications have beenexplored, and this diversity has naturally led to some contradicting observations for the cell-NSinterface and basic cell behavior. Therefore, careful, systematic studies are still needed toimprove the fundamental understanding and to optimize the NS arrays for the range ofestablished and envisioned applications, such as guiding of neurons, control of stem cell fate,intracellular electrical recordings, intracellular molecule delivery, or biosensing.The first part of this thesis is dedicated to systematic studies of cell behavior on different NSarray geometries and materials, both through theoretical modeling and experiments.We first seek to improve the fundamental understanding of the cell-NS interface bytheoretical considerations of the energy balance between the cost of membrane deformationaround the NSs and the favorable gain in adhesive contact as a cell settles into a NS array. Cellshave both been reported to deform completely into NS arrays or to stay suspended at the NS tipslike tiny fakirs, which have important implications for the possible applications. We show that,in addition to NS density, the energy difference between these two cell settling states dependshighly on the single-NS geometry. Thus, a generic cell settling prediction tool as a function ofNS diameter and length is established. We also show that the prediction depends on certain cellproperties, but that the sensitivity to changes in these is determined by the NS diameter andlength, which further underlines the importance of the NS geometry.Next, we examine the cell behavior across a range of controlled indium arsenide nanowire(NW) spacings (2-10 µm, 1-29 NWs/100 µm2) and show that the NWs generally support cellgrowth and improve adhesion, while both the morphology and cell settling height, in accordancewith the developed model, can be finely tuned through the nanowire spacing.We then extend the range of investigated NS densities by using random arrays of siliconnanocolumns (NCs), which can be fabricated with densities in the very broad range 3-700 NCs/100 µm2
Here, we observe and define three distinct cell settling regimes (fully deformed, partly deformed, and fully suspended at the NC tips), where cells behave differently
with respect to both cell morphology, detachment, mobility, actin structure and focal adhesion
formation.
Finally, we investigate the interface of cells with polymeric nanopillars (NPs) with a larger
diameter (~750 nm vs. ~100 nm for the NWs/NCs) on different NP array geometries (length,
spacing). We show that all the investigated NP array geometries support cell adhesion and
growth, and that cells form a tight interface with the NPs through a remodeling of the actin
structure, the extent of which is dependent on the NP spacing.
The second part of the thesis is dedicated to exploring the potential of NS arrays for different
cellular applications, including cell guiding, extracellular and intracellular detection.
First, in the continued study of cell behavior on polymeric NPs, cells are found to align with
the NP pattern, but the degree of alignment is strongly dependent on NP array geometry and
alignment is most efficient when cells adhere mainly to the NP tips. Therefore, we extend the
cell settling prediction tool, which was first established for NS diameters ≤500 nm, to encompass
the larger NPs. This predictive tool could then be used for the future optimization of NS arrays
for more efficient cell guiding.
Next, we show that gallium arsenide NWs interact with light in an intricate way that allows
them to be used for highly localized enhancement of fluorescence signals in close proximity
(~10 nm) to the NW surface. Using these NWs, which are wrapped tightly by the cell membrane,
we then demonstrate a 20-fold enhancement in the detection of a low-affinity interaction
between a membrane receptor and an intracellular protein.
Finally, we develop a new centrifugation-based interfacing method to promote NS penetration
through the cell membrane, so that we may also explore the potential of intracellular
applications. We show that the new interfacing method facilitates penetration of indium arsenide
NWs into the cell cytosol for 55% of the cells and that the penetrated state and cell viability
remain stable for at least 24 h. Using the established method, we then demonstrate proof-ofconcept
intracellular detection of a cytosolic protein using antibody-functionalized NWs.
Taken together, the results presented in this thesis provide important design rules for the
optimization of NS arrays for present and future applications. Furthermore, through the
establishing of new methodologies, important steps are taken towards the realization of NS
array-based extra- and intracellular live-cell sensing with a spatiotemporal resolution.
Here, we observe and define three distinct cell settling regimes (fully deformed, partly deformed, and fully suspended at the NC tips), where cells behave differently
with respect to both cell morphology, detachment, mobility, actin structure and focal adhesion
formation.
Finally, we investigate the interface of cells with polymeric nanopillars (NPs) with a larger
diameter (~750 nm vs. ~100 nm for the NWs/NCs) on different NP array geometries (length,
spacing). We show that all the investigated NP array geometries support cell adhesion and
growth, and that cells form a tight interface with the NPs through a remodeling of the actin
structure, the extent of which is dependent on the NP spacing.
The second part of the thesis is dedicated to exploring the potential of NS arrays for different
cellular applications, including cell guiding, extracellular and intracellular detection.
First, in the continued study of cell behavior on polymeric NPs, cells are found to align with
the NP pattern, but the degree of alignment is strongly dependent on NP array geometry and
alignment is most efficient when cells adhere mainly to the NP tips. Therefore, we extend the
cell settling prediction tool, which was first established for NS diameters ≤500 nm, to encompass
the larger NPs. This predictive tool could then be used for the future optimization of NS arrays
for more efficient cell guiding.
Next, we show that gallium arsenide NWs interact with light in an intricate way that allows
them to be used for highly localized enhancement of fluorescence signals in close proximity
(~10 nm) to the NW surface. Using these NWs, which are wrapped tightly by the cell membrane,
we then demonstrate a 20-fold enhancement in the detection of a low-affinity interaction
between a membrane receptor and an intracellular protein.
Finally, we develop a new centrifugation-based interfacing method to promote NS penetration
through the cell membrane, so that we may also explore the potential of intracellular
applications. We show that the new interfacing method facilitates penetration of indium arsenide
NWs into the cell cytosol for 55% of the cells and that the penetrated state and cell viability
remain stable for at least 24 h. Using the established method, we then demonstrate proof-ofconcept
intracellular detection of a cytosolic protein using antibody-functionalized NWs.
Taken together, the results presented in this thesis provide important design rules for the
optimization of NS arrays for present and future applications. Furthermore, through the
establishing of new methodologies, important steps are taken towards the realization of NS
array-based extra- and intracellular live-cell sensing with a spatiotemporal resolution.
| Original language | English |
|---|
| Publisher | Department of Chemistry, Faculty of Science, University of Copenhagen |
|---|---|
| Publication status | Published - 2016 |