Supporting information for Saleh et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0337563100

Affinity-purified monoclonal mouse antistreptavidin (IgG1) was obtained from Research Diagnostics (Flanders, NJ). Biotinylated rabbit anti-Streptococcus group A was obtained from U.S. Biological (Swampscott, MA). Unlabeled rabbit anti-Streptococcus group A was obtained from Fitzgerald Industries (Concord, MA). Extract from Streptococcus cultures was obtained from a Strep Positive Control kit made by Boule Diagnostics (Huddinge, Sweden). All other antibodies, along with soluble streptavidin powder, were obtained from Sigma. Streptavidin-coated colloids were obtained from Bangs Laboratories (Fishers, IN). Reference 470-nm sulfate-coated colloids were obtained from Interfacial Dynamics (Portland, OR). Corning no. 2 glass coverslips were obtained from Fisher Scientific. Sylgard 184 PDMS was obtained as 1.1-lb kits from the Robert McKeown Company (Branchburg, NJ).

Masters for the PDMS slabs are fabricated on quartz substrates (Mark Optics, Santa Ana, CA) in several steps. First, a 1-µm-wide line of photoresist (AZ 5214E IR, Clariant, Somerville, NJ) is patterned on the substrate. A reactive ion etch is then used to remove ≈1 µm of quartz surrounding the line, and the remnant photoresist is dissolved. This subsequently forms the raised line, ≈1 µm × 1 µm in cross section, that later is cast into PDMS as the pore. The negative reservoirs are formed by photolithographically patterning 7-µm-thick SU-8 photoresist (Microchem, Newton, MA) on the substrate. PDMS is mixed at a 10:1 resin/catalyst ratio, poured over each master, degassed, and cured for at least 24 h at 80°C to insure stable mechanical properties. Immediately before measurement, PDMS slabs with the embedded pore and reservoirs are cut from each master (masters are then cleaned and can be reused indefinitely). Access holes are cut into each slab by coring with a needle. Each slab is cleaned by a 5-min wash in a solution of 1% Tween 20 (Sigma) followed by extensive rinsing in deionized water.

The electrodes are patterned on glass coverslips by photolithography in AZ 3318D resist (Clariant) and formed by depositing 5 nm of titanium and 25 nm of platinum in an electron-beam evaporator. Immediately before sealing to the PDMS, the coverslips are cleaned in a dilute RCA SC1 cleaning solution (10:1:1 H₂O/H₂O₂/NH₄OH heated to boiling for ≈10 min) and rinsed in copious amounts of deionized water.

Cleaned PDMS slabs are treated in an air plasma to create a hydrophilic surface (1) and strengthen the bond to the glass substrate (2). A 5-µl drop of deionized water is placed on top of a clean coverslip, and then the clean, plasma-treated PDMS slab is placed on the coverslip. The water allows the slab to slide on top of the coverslip for alignment of the pore between the electrodes. Once aligned, the PDMS/coverslip assembly is placed on a hot plate and subjected to a 10-min bake ramped from 50°C to 150°C. This bake serves to drive off the water and induce a strong bond between the PDMS and coverslip. The device is now ready for addition of solution and measurement.

Once the device is wet with solution, pressure is applied to either reservoir by using a commercial microfluidic pump (Fluidigm, South San Francisco, CA). The pump is connected to the device through 0.020-inch i.d./ 0.060-inch o.d.Tygon tubing (Fisher Scientific) that is inserted into the access holes in the PDMS slab. A pressure of 7–14 kPa (1–2 psi) is typically applied during measurements. Clogs in the pore occur occasionally and are cleared by application of higher pressures (up to 20 psi), without any effect on the PDMS/coverslip seal.

Electrical current is measured at constant applied DC voltage (typically 0.2–0.4 V) with a four-point measurement by using a homemade feedback circuit, as described (3). The current is passed into a preamplifier that performs a low pass filter at 0.03 ms in rise time. The resulting output is connected to a data acquisition board in a computer for data sampling at 50 kHz.

1. Fakes, D. W., Davies, M. C., Brown, A. & Newton, J. M. (1988) Surface Interface Anal. 13, 233–236.

2. Chaudhury, M. K. & Whitesides, G. M. (1991) Langmuir 7, 1013–1025.

3. Saleh, O. A. & Sohn, L. L. (2001) Rev. Sci. Instrum. 72, 4449–4451.