Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules

Summary

We present a procedure for forming a poly(ethylene glycol) self-assembled monolayer (PEG-SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single step and prevents biofouling on silicon and gold surfaces. Electrophoresis is then used for patterning biomolecules down to the nanoscale.

Abstract

We present a procedure for forming a poly(ethylene glycol) (PEG) trimethoxysilane self-assembled monolayer (SAM) on a silicon substrate with gold microelectrodes. The PEG-SAM is formed in a single assembly step and prevents biofouling on silicon and gold surfaces. The SAM is used to coat microelectrodes patterned with standard, positive-tone lithography. Using the microtubule as an example, we apply a DC voltage to induce electrophoretic migration to the SAM-coated electrode in a reversible manner. A flow chamber is used for imaging the electrophoretic migration and microtubule patterning in situ using epifluorescence microscopy. This method is generally applicable to biomolecule patterning, as it employs electrophoresis to immobilize target molecules and thus does not require specific molecular interactions. Further, it avoids problems encountered when attempting to pattern the SAM molecules directly using lithographic techniques. The compatibility with electron beam lithography allows this method to be used to pattern biomolecules at the nanoscale.

Protocol

Preparation of reagents

Prepare PIPES buffer (80 mM PIPES, 1mM MgCl₂, 1mM EGTA, adjusted to pH 6.8 with KOH). Aliquot and store glucose oxidase, catalase and glucose at -70°C and taxol at -20°C according to existing protocols.^1,2 Aliquot and store tubulin at -70°C according to the instructions included by the supplier.

Pattern Au microelectrodes using lithography

1. Cut silicon wafers into 1 cm ´ 1.5 cm substrates using .......

Discussion

The migration of MTs is easily visualized using fluorescence microscopy because of their brightness and the low background fluorescence. The method is generally applicable to biomolecule patterning, as it only requires that the biomolecules be induced to carry a net charge by suitable adjustment of the buffer pH. Electroosmosis is avoided in this setup because there are no charged surfaces such as the silica walls used in capillary electrophoresis.

Several modifications of this method are pos.......

Materials

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
2-[methoxypoly-(ethyleneoxy)propyl]-trimethoxysilane (PEG-silane)		Gelest, Inc.	SIM6492.7	6-9 PEG units; molecular weight 450-600 g mol-1
2-mercaptoethanol		Sigma	M7522
Acetone		EMD Chemicals	AX0120-8	ACS grade
Catalase		Calbiochem	219001-5MU
Chlorobenzene		EMD Chemicals	MK441908	ACS grade
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA)		Calbiochem	324626
Glucose		EMD Chemicals	DX0145-1
Glucose oxidase		MP Biomedicals	100330
Guanosine-5’-[(α,Β)-methyleno]triphosphate, Sodium salt (GMPCPP)		Jena Biosciences, GmbH	NU-405s
Guanosine-5’-triphosphate		Sigma	G8752
Isopropanol		EMD Chemicals	PX1835-5	ACS grade
Methyl isobutyl ketone (MIBK)		Fisher Scientific	AC12739-0010
Paclitaxel (taxol)		Calbiochem	580555
Piperazine-N,N''-bis(2-ethanesulfonic acid) (PIPES)		Sigma	P1851
Polymethylmethacrylate (PMMA)		Brewer Science, Inc.	950K	molecular weight 950K
Rhodamine tubulin		Cytoskeleton, Inc.	T331M	from bovine brain 99% pure, lyophilized
Toluene		EMD Chemicals	TX0735-5	ACS grade
Tubulin		Cytoskeleton, Inc.	T238	from bovine brain 99% pure, lyophilized

No. 1 borosilicate glass coverslips (22 X 50 mm) were purchased from VWR. Polished electronic grade p-type <111> silicon wafers were purchased from Addison Engineering, Inc. High-purity silver paint was obtained from Structure Probe, Inc. (Catalog # 5001 AB). Insulated fine gauge copper magnet wire (~ 0.08 mm (0.003”) diameter) can be obtained from electronic supply companies.