Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Summary

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Abstract

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.¹ While microcontact printing can be employed to directly print a large number of molecules, including proteins,² DNA,³ and silanes,⁴ the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.⁵ This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.

The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.⁶ These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior⁷ to the creation of microelectronics.⁸

While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,⁹ patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.¹⁰

Protocol

1. Preparation of the Patterned Master (Figure 1)

1. Center the silicon wafer on the spin-coater and rinse the wafer with acetone during the initial step of the two-cycle spin program in Table 1. The acetone will evaporate during the second step of the spin program leaving a clean, dry wafer.
2. Apply approximately 1 mL AZ9245 photoresist/in (in diameter) to the wafer and spin-coat using the conditions described in Table 1.
3. Soft-bake the photoresist-coated wafer at 110°C for 2 m using a high-uniformity hotplate.
4. Photopattern the substrate using either a direct-write photolithography system or a mask aligner system and appropriate mask.....

Discussion

A number of issues can arise in the lithographic production of the master used for PDMS stamp creation. Underexposure of the resist-coated wafer results in hazy and indistinct patterns and overexposure of the resist-coated wafer results in enlarged or missing features. In general, masters with large feature sizes (>10 μm) are relatively easy to pattern and develop, while masters with smaller features can require extensive optimization of photopatterning and development parameters (beyond the parameters recommended .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon wafer	Wafer Reclaim Services		2 inch
Spin coater/hot plate	Brewer Science, Inc.	Cee 200CB Spin-Bake System
AZ9245 Photoresist	Mays Chemical Company	105880034-1160
Direct-write photolithography system	Microtech s.r.l.	LW325 LaserWriter System
Mask Aligner	HTG	3HR
AZ 400K Developer	Mays Chemical Company	105880018-1160
Sylgard 182 Silicone Elastomer Kit	Dow Corning
25 mm no. 1 round glass coverslips	VWR international	16004-310
Plasma Oxidizer	Diener	Femto
Titanium piecesKamis	Incorporated		99.95% pure
Gold pellets	Kamis Incorporated		99.999% pure
Electron-beam evaporator	Kurt J. Lesker	PVD 75 Thin Film Deposition System	with electron-beam accessory
Hexadecanethiol	Alfa Aesar	A11362
1-mercaptoundec-11-yl)tetra(ethyleneglycol)	Sigma-Aldrich	674508
Ethanol	Pharmco-AAPER	111000200	200 proof, absolute
Parafilm	VWR international	52858-000
DPBS	VWR international	4500-434	Without calcium and magnesium
Mouse Laminin I	VWR international	95036-762
Human Plasma Fibronectin	Invitrogen	33016-015
AlexaFluor® 647 carboxylic acid, succinimidyl ester	Invitrogen	A-20006
MitoTracker Red 580	Invitrogen	M22425
AlexaFluor® 350 carboxylic acid, succinimidyl ester	Invitrogen	A-10168
Anti-laminin antibody	Fisher Scientific	AB2034MI