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In This Article

  • Summary
  • Abstract
  • Protocol
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

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).

....

Materials

Material NameTypeCompanyCatalogue NumberComment
NameCompanyCatalog NumberComments
2-[methoxypoly-(ethyleneoxy)propyl]-trimethoxysilane (PEG-silane) Gelest, Inc.SIM6492.76-9 PEG units; molecular weight 450-600 g mol-1
2-mercaptoethanol SigmaM7522 
Acetone EMD ChemicalsAX0120-8ACS grade
Catalase Calbiochem219001-5MU 
Chlorobenzene EMD ChemicalsMK441908ACS grade
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) Calbiochem324626 
Glucose EMD ChemicalsDX0145-1 
Glucose oxidase MP Biomedicals100330 
Guanosine-5’-[(α,Β)-methyleno]triphosphate, Sodium salt (GMPCPP) Jena Biosciences, GmbHNU-405s 
Guanosine-5’-triphosphate SigmaG8752 
Isopropanol EMD ChemicalsPX1835-5ACS grade
Methyl isobutyl ketone (MIBK) Fisher ScientificAC12739-0010 
Paclitaxel (taxol) Calbiochem580555 
Piperazine-N,N''-bis(2-ethanesulfonic acid) (PIPES) SigmaP1851 
Polymethylmethacrylate (PMMA) Brewer Science, Inc.950Kmolecular weight 950K
Rhodamine tubulin Cytoskeleton, Inc.T331Mfrom bovine brain 99% pure, lyophilized
Toluene EMD ChemicalsTX0735-5ACS grade
Tubulin Cytoskeleton, Inc.T238from 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

  1. Rai-Choudhury, P. . Handbook of Microlithography, Micromachining, and Microfabrication: Microlithography. , (1997).
....

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