Materials and Methods Visually Apparent SAMs

Synthesis of alkanethiols and peptides. The undecane thiols terminated with tetra(ethylene

glycol), hydroquinone–tetra(ethylene glycol), and of RGD–oxyamine were synthesized as reported previously.7, 18, 19

Electrochemistry. All electrochemical measurements were made by using a bioanalytical

Systems Epsilon potentiostat. An Ag/AgCl electrode served as the reference, the gold monolayer acted as the working electrode, and a platinum wire served as the counter electrode. The electrolyte was HClO4 (1 M) and the scan rate was 100 mVs-1. All measurements were made in a standard electrochemical cell.

Microfabrication. The microchips were fabricated by using soft lithography.20 Patterns were

fabricated by using masks drawn in Adobe Illustrator CS3 and photoplotted by Pageworks onto transparencies. The SU-850 (MicroChem, Newtown, USA) was patterned by using the manufacturer’s directions, and a 100 mm channel depth was obtained with these masks. Slygard 184 (Dow Corning) was cast onto the mold by using a 1:10 ratio of Pt curing catalyst to elastomer (w/w). The prepolymer was degassed for 15 min and then poured over the mold.

The prepolymer was then cured for 1 h at 75oC. The PDMS was removed from the master and access holes were punched into the PDMS to allow fluid flow.

Preparation of monolayers. Gold substrates were prepared by electron beam deposition of

titanium (6 nm) and gold (24 nm) on 24 mm 100 mm glass microscope slides. The slides were cut into 1 cm2 pieces and washed with absolute ethanol before use.

Chemical gold etch. A PDMS microfluidic stamp was reversibly sealed to a gold substrate.

To chemically etch the gold surface, a solution containing potassium iodide (18 mM) and iodine (4.3 mM) was flowed into microchannels for various lengths of time (Figure 2.2) to partially etch the gold layer. By controlling the duration of the etch conditions, it was possible to control the amount of gold etched. Once the gold had been etched, flowing water and then ethanol (30 s each) were used to clean the microchannels.

SAM Formation. For the patterned and electroactive surface characterization, a 1 mM

solution of HQ-EG4 in ethanol was flowed into the channels for 30 s to install a SAM on the etched regions. The microfluidic device was then removed and the remaining bare gold regions were backfilled with EG4 for 10 min. Cyclic voltammetry was used to determine the density of electroactive HQ-EG4 on the etched regions. For cell biology studies, a mixed SAM that contained HQ-EG4 (10%) and EG4 (90%) was flowed through the microchannels (1 mM total, 1 min). The microfluidic cassette was then removed, and the remaining bare gold regions were backfilled with EG4 (1 mM) for 8 h. The surface was electrochemically oxidized and the BQ surface was treated with amino-oxy-RGD (1 mM, 2 h) to install the peptide on the surface for subsequent biospecific cell adhesion and migration studies.

Oxyamine coupling reaction. The surface was activated with the application of 750 mV for

34

acetic acid (250 mM) was added and incubated on the surface for 2 h. For cell experiments the same activating conditions were used but RGD–oxyamine peptide was immobilized to the surface (1 mM, 2 h).

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