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.
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
Prepare the SAM
Fabricate the counterelectrode
In the same deposition chamber setup used for microelectrode deposition, make a counterelectrode by depositing 1 nm of Cr, followed by 4 nm of Au onto a 22 x 50 mm No. 1 or No. 0 glass coverslip. The counterelectrode should be nearly transparent with a light gray or pink tint. Store the counterelectrode coated-side-up in a Petri dish to keep it clean.
Polymerize tubulin into MTs
Pattern MTs using electrophoresis
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 possible. Sealing the ends of the flow chamber is critical to observe reversibility because evaporation from the open ends causes bulk fluid flow and undesirable MT migration. Oxidation of the silicon wafer prior to lithographic patterning will prevent conduction through the substrate, allowing for multiple electrodes with independent potentials to be used simultaneously.
The authors have nothing to disclose.
We acknowledge Melissa Grunlan for helpful discussions on SAM formation. This research was supported in part by the Robert A. Welch Foundation (A-1585).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
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.