12. Pipet 1.0 mL of PBSTB into each reservoir and restart the pump at a 1-mL/min flow rate. Allow each channel to rinse with PBSTB and finally evacuate.
13. Remove all fluidics connections from the patterning template and disassemble patterning template sandwich.
14. Place patterned slide into blocking solution and incubate for 30 min.
15. Dry the patterned, blocked slides under nitrogen stream. Slides can be stored at 4⬚C for as long as 5 mo (11).
tion reaction. Therefore, antibody stocks in these buffers must be subjected to a buffer exchange process, preferably with PBS.
1. Dilute 1 mg of antibody with PBS to a final protein concentration of 1 to 2 mg/mL (use a 1.5- or 1-mL centrifuge tube).
2. Add 1/9 volume of 0.5 M borate buffer, pH 8.5, such that the final borate buffer concentration is 50 mM.
3. Dissolve 1 vial of Cy5-NHS-ester in 50 µL of DMSO immediately before adding to the antibody (see Note 10).
4. Add 15 µL of Cy5/DMSO mix to the antibody.
5. Wrap the vial with aluminum foil and incubate at room temperature for 30 min while rocking (see Note 11).
6. Pipet mix onto a 25 mL of Bio-Gel P-10 column that has been pre-equilibrated in PBS (see Note 4). Allow the sample to soak into gel and rinse top of gel and sides of column with PBS.
7. Add PBS onto of the top of gel, collect and save the first blue fraction. Store in the dark.
8. Take appropriate volume of the eluted first blue fraction and dilute with PBS (typi- cally 10× dilution).
9. Take the absorbances at 280 nm and 650 nm. Determine the concentration of the antibody and the dye to protein ratio (see Note 12). Store Cy5-labeled antibody in the dark at 4⬚C.
3.3.2. Preparation of Food Samples
Each food is prepared differently depending on its texture, with the goal of making it homogenous enough to flow through the syringes and tubing at a 0.1 mL/min flow rate. Protocols detailed are based on those used by the US Food and Drug Administration, the Center for Food Safety and Applied Nutrition, and the US Department of Agriculture (25–27).
3.3.2.1. HAM, GROUND BEEF, PORK SAUSAGE, CANTALOUPE 1. Weigh out several aliquots of each food (1–10 g).
2. Add equal volume of PBSTB containing various concentrations of the analyte.
3. Place the food–buffer mixture (with or without analyte) into a Waring blender and homogenize at high speed for 2 min.
4. Transfer the homogenate samples into centrifuge tubes (15-mL volume) and cen- trifuge at 3000g for 10 min.
5. Analyze the supernatant.
3.3.2.2. CHICKEN CARCASS WASH
1. Place a fresh chicken carcass and 100 mL of PBS containing 1 mg/mL of BSA in a resealable bag (e.g., Ziplock®).
2. Seal the bag and incubate for 2 h at room temperature on a rocking platform.
3. Remove the liquid (carcass wash) and spike aliquots with analyte (see Note 13).
4. Analyze without further treatment.
3.3.2.3. EGG
1. Blend whole eggs until in liquid form and aliquot into the appropriate volumes.
2. Added equal volume of PBSTB containing various concentrations of analyte.
3. Transfer the egg–PBSTB mix into a Waring blender and mix on high speed for 2 min.
4. Analyze the diluted homogenate without further treatment.
3.3.3. Assay Protocols
Sample analysis is performed on the “capture” antibody-patterned slides using the PDMS assay template and mounting manifolds described in Subhead- ing 3.2.2. However, for assays, the upper piece of the assay-mounting manifold has the two openings for needle insertion perpendicular to those for patterning.
1. Place patterned slide face-up into slot of lower assay manifold (same manifold used for patterning).
2. Place PDMS assay template on the surface of patterned slides, with channels facing the slide and orthogonal to the stripes of the immobilized “capture” anti- body.
3. Place upper assay manifold on top of the PDMS and tighten into place with screws.
4. Insert needle with an open barrel into one end of each PDMS channel (inlet; see Fig. 4).
5. Insert needle attached to tubing of a (multichannel) peristaltic pump into the other end of each PDMS channels (outlet; see Note 14).
6. Pipet 1.0 mL of PBSTB into each syringe barrel reservoir and start the pump at a 1 mL/min flow rate. After the PBSTB has flowed through each channel and res- ervoir is empty, allow each channel to fill with air and stop the pump.
7. Pipet 0.8 mL of the prepared food sample containing the various concentrations of analytes into the syringe reservoirs. Include a buffer blank (0.8 mL of PBSTB).
Restart the pump at a 0.1 mL/min flow rate (see Note 15).
8. Allow the assay to run for 15 min then empty the channels. Allow them to fill with air and stop the pump.
Fig. 4. Schematic diagram of the assembled patterned slide and fluidics during as- say. The upper and lower manifolds are not shown.
9. Pipet 1 mL of PBSTB into the syringe reservoir and start the pump at a 1 mL/min flow rate. After approx 1 min, allow each channel to fill with air and stop the pump (see Note 16).
10. Pipet 0.4 mL of the “tracer” antibody into each reservoir, start the pump at a 0.1 mL/min flow rate. After the “tracer” has flowed through each channel and reser- voir is empty, allow each channel to fill with air and stop the pump.
11. Pipet 1 mL of PBSTB into the syringe reservoir and start the pump at a 1 mL/min flow rate. After approx 1 min, allow each channel to fill with air and stop the pump.
12. Remove all fluidics connections from the assay template and disassemble assay template.
13. Rinse the entire slide with water and dry under a stream of nitrogen.
14. Image slide immediately or store in the dark for up to 24 h for later imaging.