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.
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Method Article
Antifouling Self-assembled Monolayers on Microelectrodes for Patterning Biomolecules
In This Article
Summary
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
- Cut silicon wafers into 1 cm ´ 1.5 cm substrates using a scribe or wafer cutter. Clean the substrates by placing them in a beaker with enough acetone to cover them and sonicate for 5 minutes. Without allowing the substrates....
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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.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge Melissa Grunlan for helpful discussions on SAM formation. This research was supported in part by the Robert A. Welch Foundation (A-1585).
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Materials
| 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-mercapt–thanol | Sigma-Aldrich | M7522 | |
| Acetone | EMD Millipore | AX0120-8 | ACS grade |
| Catalase | Calbiochem | 219001-5MU | |
| Chlorobenzene | EMD Millipore | MK441908 | ACS grade |
| Ethylene glycol-bis(2-amin–thylether)-N,N,N’,N’-tetraacetic acid (EGTA) | Calbiochem | 324626 | |
| Glucose | EMD Millipore | 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-Aldrich | G8752 | |
| Isopropanol | EMD Millipore | 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-Aldrich | P1851 | |
| Polymethylmethacrylate (PMMA) | Brewer Science, Inc. | 950K | molecular weight 950K |
| Rhodamine tubulin | Cytoskeleton, Inc. | T331M | from bovine brain 99% pure, lyophilized |
| Toluene | EMD Millipore | 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. |
References
- Tubulin Polymerization with GTP/GMPCPP/Taxol [Internet]. , Harvard University. Cambridge, Ma. Available from: http://mitchison.med.harvard.edu/protocols/poly.html (2009).
- Flow Cell Assays with Microtubules: Motility/Dynamics [Internet]. , Harvard University. Cambridge, Ma. Available from: http://mitchison.med.harvard.edu/protocols/flowcell.html (2009).
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