Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Summary

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Abstract

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

Introduction

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation^1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls^2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron^2,11,12, muscle¹³, and bone^3,4. In addition, be....

Protocol

1. Replication of PDMS Molds from an EBL Mold

1. Fabricate silicon mold²⁹
   1. Spin coat 200 μl polymethyl methacrylate (PMMA) solution on a 1 × 1 cm silicon (Si) substrate at 2,500 rpm for 1 min to form a thin film.
   2. Bake the PMMA film on the Si substrate at 180 °C for 2 min.
   3. Write the designed nanopattern on the PMMA film by using a focused electron beam at an area dose of 300 µC/cm².
   4. Develop the PMMA nanopattern in developer for 80 sec.
   5. Deposit the PMMA nanopattern with a nickel layer of 50 nm in thickness using an E-beam evaporator at an output voltage of 10 k....

Results

The stitch technique can generate a large area of nanopatterned substrates with high fidelity. Figure 1a and 1b display the large area of nanopatterns transferred from the stitched PDMS mold to PS plate and PS thin film on a Si substrate, respectively. The comparison between the original EBL-written mold (Figure 1c) and the final PDMS working substrate (Figure 1d) confirms that the EBL-written nanopatterns can be faithful.......

Discussion

We present a simple, affordable, yet versatile method to generate a large area of nanopatterned substrate. To faithfully expand highly defined nanopatterns, great attention should be paid to several critical steps. The first one is to trim the multiple PDMS molds. Unpatterned areas of the PDMS molds need to be removed. Additionally, the sidewalls of the molds should be cut vertically as perfect as possible to minimize the gaps between the molds. Collectively, the portion of unpatterned areas in the final stitch mold can .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
JEOL field emission SEM	JEOL	JSM-7600F	EBL
E-beam evaporator	Kurt J. Lesker	Model: LAB 18 e-beam evaporator	nickel deposition
Trion Minilock III ICP/RIE	Trion technology	Model: Minilock-phantom III
Press machine	PHI Hydraulic Press	Molde: SQ-230H
Spin coater	Laurell Technologies	Modle: WS-400A-6NPP-LITE
CO2 critical dryer	Tousimis	Modle: Autosamdri-815
Silicon wafer	University Wafer	1080
Aluminum plates	McMaster-carr	9057K123
Teflon sheets	McMaster-carr	8711K92
100 mm Petri dish	FALCON	353003
60 mm Petri dish	FALCON	353004
Glass coverslip	Fisher Scientific	12-542-B
Glass slide	Fisher Scientific	12-550-34
Disposable weighing boats	Fisher Scientific	13-735-743
Glass desiccator	Fisher Scientific	02-913-360
Plastic desiccator	Bel-Art Products	F42025-000
Hotplate	Fisher Scientific	1110049SH
Tweezer	Ted Pella, inc.	5726
Blade	Fisher Scientific	S17302
Metal blocks	McMaster-carr
Punch	Brettuns Village Leather Craft Supplies	Arch punch
Poly(methyl methacrylate)	MicroChem	495 PMMA A4
PDMS	Dow Corning	Sylgard 184 kit
Polystyrene	Dow Chemical	Styron 685D
1H,1H,2H,2H-perfluorooctylmethyldichlorosilane	Oakwood Chemical	7142
Developer	MicroChem	MIBK/IPA at 1: 3 ratio
Remover	MicroChem	Remover PG
Ethanol	Fisher Scientific	BP2818500
Toluene	Fisher Scientific	T324-500
Phosphate buffered saline	Sigma Aldrich	D8537
Dulbecco’s modified eagle medium	Sigma Aldrich	D5796
Fetal bovine serum	Atlanta Biologicals	S11550
Paraformaldehyde	Electron Microsopy Science	15712-S
Glutaraldehyde	Fisher Chemical	G151-1
Fibronectin	Corning	356008
A549 cells	ATCC	ATCC CCL-185