Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

1. Substrate Preparation

1. Coat layered silicon-SiO₂ substrates with self-adsorbing copoly(DMA-NAS-MAPS):
   1. Copolymer synthesis, chemical structure, and coating process are published in: G. Pirri, F. Damin, M. Chiari, E. Bontempi, L.E. Depero. Characterization of A Polymeric Adsorbed Coating for DNA Microarray Glass Slides. Anal. Chem. 2004, 76, 1352-58.
   2. Briefly, prepare polymer solution by adding 100 mg of polymer to 5 mL of deionized (DI) water and add 5 mL of 40% saturated ammonium sulfate ((NH₄)2SO₄) solution to reach a final concentration of 0.92 M.
   3. Submerge chips in solution for 30 min on a shaker and then rinse thoroughly with DI water. Dry thoroughly with Argon/N₂ gas.
   4. Bake chips at 80 °C for 15 min.
2. Store polymer-coated substrates/chips in a dry environment (vacuum desiccator) for up to 3 months until probe spotting procedure.

2. Preparation of Probe Array: Antibodies, antigens, ss/dsDNA, RNA, etc.

1. Dilute probe(s) to appropriate concentration in desired buffer. This step can be vary considerably, but a typical experiment utilizes antigen or IgG at a concentration of 0.5 mg/mL (range of 0.1 -1 mg/mL) in phosphate buffered saline (PBS) at pH≈7.4.
2. Place solutions in a 96- or 384-well plate (or standard source plate for the spotter being used)
3. Setup spotting parameters for desired printed array: determine the appropriate spotting parameters for the surface and solutions being used (dwell times, approach speeds, etc.). Determine the number of replicates of each condition per grid, the grid layout, the number of replicate grids, and the desired spotting location on the chip. Place substrates and source plate in the appropriate locations, check to make sure waste and supply bottles are ready, and begin printing run.
4. After spotting is finished place substrates in a high humidity environment overnight (4-18 hours) to allow immobilization and deactivation process to proceed.
5. Wash substrates: place them in the following solutions for three minutes for three separate washes: PBS with 0.1% Tween (PBST), PBS, and finally deionized (DI) water. Depending on the type of experiment (wet vs. dry), dry the chip thoroughly with Argon gas and begin incubation or place the chip in a flow chamber and assemble.
6. Deactivate remaining NHS groups on copolymer surface by submerging the chips in a 50 mM solution of ethanolamine with the pH adjusted to 7.4 for 30 minutes while on a shaker plate. A primary amine-containing compound such as hydroxylamine or Tris can also be used, as long the prepared solution is safe to use with proteins. Thoroughly rinse the deactivation solution using PBS then DI water.
7. Optional step (depending on the surface chemistry being employed): Block surface using a solution of BSA, casein, or rehydrated milk for 30 minutes. For the current polymeric surface chemistry that we use, we do not perform this step as the polymer backbone acts to prevent non-specific protein binding.

3. Data Acquisition and Incubation Procedure: Endpoint format

1. Make a solution of the target of interest in the desired buffer. Here, it will be a solution of the appropriate concentration of Anti-BSA in PBS (ex: make 1-10 mL of a 10 ng/mL solution of Anti-BSA in PBS). Also, make sure to have an equivalent amount of buffer (without target) for the negative control incubation.
2. Take a silicon mirror scan for normalizing all subsequently collected data.
3. Scan the prepared probe array with the IRIS system prior to the incubation step to determine the initial optical height (mass) at each spot. This will allow for proper analysis of changes in mass on the surface, i.e. the level of binding, after the incubation. Here, a few parameters will be adjusted for the current experiment, such as exposure time, field of view, prospering focusing, etc.
4. Submerge chip(s) in prepared solution for 1 hour on a shaker plate. The incubation time can vary considerably depending on the level of agitation, the concentration of target being detected, the transport characteristics of the flow chamber (if a real-time detection mode is being used), the affinities of the target-probe pair, etc.
5. After the incubation, repeat the wash procedure described in step 2.5)
6. Scan the same probe array using the IRIS system and obtain post-incubation images for pre-incubation comparison.
7. Repeat this process for different incubation steps if necessary, for example in a sandwich assay format where a secondary antibody is being used.

4. Data Analysis

1. Fit the collected images for each IRIS scan (sequence of intensity images), using the lab-developed software to produce an image of the probe array containing optical thickness information. A quick qualitative analysis of binding can be performed by subtracting (registered) post- and pre-incubation images. Binding will appear as a change in intensity at those spots where mass changes were observed. For quantitative analysis, determine mass densities of each spot compared to the background by averaging optical thickness information for each pixel within a spot and an annulus outside of the spot and making a direct comparison of these two areas.

5. Representative Results:

Figure 1 shows an example schematic of the layered Si-SiO₂ IRIS substrate spotted with a representative antibody array. Each antibody ensemble is spotted in replicate with specificity for a different epitope targeting different proteins. Two different whole viruses and a viral protein are represented as example targets. Negative control antibodies depend on the assay and can be non-specific and/or virus-specific.

Figure 2 shows the binding of human serum albumin (HSA)-specific antibodies to an array of spotted HSA and rabbit IgG (control) spots. As seen here, after fitting and determination of the optical thicknesses for the pre- and post-incubation images, a difference image for the binding array can be created to qualitatively assess binding. Quantitative analysis reveals that a 2.05 and 0.13 nanometer mean optical height change was observed for the HSA and rabbit IgG spots, respectively, for an anti-HSA incubation concentration of 500 ng/mL.

Figure 3 shows an IRIS measurement of Fur protein binding dependence on protein concentration and dsDNA oligomer length. The top graph shows absolute optical height measurements for initial immobilized oligomer probe heights and post-incubation Fur binding. Increasing concentrations of Fur (50, 100, 200 nM) resulted in increased binding to DNA probes. The length of the oligomers also impacts Fur binding with longer sequences resulting in more binding. Here, error bars represent one standard deviation from the mean. The bottom plot shows calculated Fur protein dimer binding per dsDNA strand. Dimer binding was significantly increased at a Fur protein concentration of 200 nM suggesting a high-level of non-specific binding.

Figure 1. Schematic of an example probe array normally used in an experiment to detect specific viruses and viral components in a multiplexed fashion with the IRIS system.

Figure 2. Post-incubation difference image for binding of human serum albumin-specific antibodies to spotted HSA collected with this label-free system at a concentration of 500 ng/mL. The biomaterial mass density for each spot is determined by averaging optical thickness information within a spot and then comparing this with the average of an annulus around the spot representing the background.

Figure 3. Plot of binding data for Fur protein interactions with spotted double-stranded oligomers with a known consensus sequence based on oligomer length, location of the consensus sequence within the oligomer, and Fur protein concentration. Information about the absolute amount of Fur protein bound to each spotted sequence (top) can be used to estimate the number of Fur dimers bound to each type of oligomer (bottom).