The overall goal of the following experiment is to quantify the binding of antibodies to surface immobilized antigens with a sensitive and label-free sensing platform termed Iris. This is achieved by first preparing an array of test and control probes on a chemically functionalized uniform silicon dioxide surface to capture targeted proteins from solution. The immobilized probes can be antibodies or antigens, DNA or RNA or any other type of macromolecule that is compatible with the functionalized, polymeric, or sane surface chemistries.
The prepared microarray is then incubated with a sample containing the proteins of interest, which allows binding to occur under conditions specified by the researcher. Next, the entire sensor array is imaged at different wavelengths of light. With the IRIS system, the reflected intensities for each pixel are processed with custom coded algorithms.
In order to quantify the amount of biomaterial bound to each test or control condition, post incubation results are obtained that show effective detection and absolute quantification of antibodies in a complex mixture based on the interferometric label-free measurements of this system Building on more than a decade of research and development on optical interference and resin cavity devices, we develop this new method that could overcome many of the confounding issues that we encountered with our previous label Free biosensor systems. Visual demonstration of this method is critical as the data collection steps require the correct normalization and quality control procedures are followed. To ensure accurate and repeatable data acquisition.
To co lay its silicon dioxide substrates with selfs absorbing copo dmma NAS maps first, repair the polymer solution by adding 100 milligrams of the polymer to five milliliters of deionized water. Then add five milliliters of 40%saturated ammonium sulfate for a final ammonium sulfate concentration of 0.92 molar. Submerge the chips in the polymer solution for 30 minutes on a shaker following incubation.
Rinse the chips thoroughly with deionized water, dry the chips with argon or nitrogen gas, and then bake them at 80 degrees Celsius for 15 minutes. Store the polymer coated substrate chips in a dry environment such as a vacuum desiccate for up to two to three months until the probe spotting procedure to dilute each probe. First, determine the correct volume of the probe stock solution and buffer to achieve the desired final concentration.
A typical experiment utilizes antigen or IgG as a concentration of 0.5 milligrams per milliliter in phosphate buffered saline at the calculated amount of each probe stock solution to small eend orph tubes containing the desired amount of PBS buffer, mixing the solutions. Well then pipette the probe solutions into a 96 well low protein binding plate, which will be used as the source plate for the spotter. Next, set up the spotting parameters for the printed array by determining the number of replicates of each condition per grid.
The grid layouts the number of replicate grids and the desired spotting location on the chip. Place the substrate and the source plate in the spotter and then check the waste and supply bottles. Begin the printing run.
After spotting is finished, place the substrate in a high humidity environment overnight to allow the immobilization and deactivation process to proceed following incubation. Wash the substrates with agitation by first placing the chip in PBS with 0.1%tween for three minutes, three separate times. Follow this with three three minute incubations in PBS and then in deionized water.
If performing a dry measurement, dry the chip thoroughly with argon gas. Deactivate the remaining NHS groups on the copolymer surface by submerging the chips in a 50 millimolar solution of ethanol amine with the pH adjusted to 7.4 for 30 minutes while on the shaker plate. Thoroughly rinse the ethanol amine using PBS and then deionized water prior to the start of the incubation procedure.
Prepare a solution of the appropriate concentration of target protein in PBS for this demonstration. 500 nanograms per milliliter of anti-human serum albumin is used include an equivalent volume of buffer without target for the negative control incubation. The iris system consists of a few basic elements, such as an illumination source, optical components for properly illuminating the sensor probe array, a camera for recording reflected light intensities, and a stage where the sample is placed for measurements.
Begin with a silicon mirror scan of a bare clean silicon chip to normalize for all subsequently collected data prior to performing the scan. Prepare the iris system by adjusting parameters for the experiment. This includes measuring the histogram of light intensities, which the camera records, and setting the exposure time for optimal intensities.
After the normalization scan, load the prepared probe array into the iris system and perform the setup as before, set the focus to correctly image the substrate surface. Make sure that the sample stage is level by inspecting the recorded histograms for reflected light intensities. Finally, choose a file name to save the data under.
Initialize the software to scan the array, which illuminates the surface at different wavelengths many times and averages the collected images. The initial optical high to teach spot is determined, allowing for proper analysis of changes in mass on the surface after the incubation following the initial scan, submerge the chip in the prepared incubation solution for one hour on a shaker plate. The incubation time can vary considerably depending on the level of vegetation, the concentration of target being detected, and the affinities of the target probe pair.
After the incubation, repeat the wash procedure as previously performed. Scan the same pro array using the iris system to obtain post incubation images for pre incubation comparison. This process can be repeated for different incubation steps if necessary.
For example, in a sandwich assay format where a secondary antibody is being used, fit the collected images for each iris scan using commercially developed software to produce an image of the proby containing optical thickness formation. A quick qualitative analysis of binding can be performed by subtracting 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 for an annulus outside of the spot. Then make a direct comparison of these two areas shown here. As an example, schematic of the layered silicon, silicon dioxide iris substrate spotted with a representative antibody array.
Each antibody ensemble is spotted in replicate with specificity for a different epitope, targeting a different protein. 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.
Here is an example of qualitative results for binding of human serum albumin specific antibodies to an array of spotted HSA and rabbit IgG control spots as seen here. After fitting and determination of the optical thickness for the pre and post incubation images, a difference image for the binding array can be created to rapidly assess binding. More hor 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 nanograms per milliliter.
This bar graph represents an example of the quantitative data iris measurements can provide. In this example, fur protein binding dependence on protein concentration and double stranded all ligament length is presented. This bar graph shows the absolute optical height measurements for initial immobilized all ligament prop heights and post incubation for binding.
Increasing concentrations of fur protein to 5, 100 and 200 nanomolar resulted in increased binding to DNA probes. Here, the calculated fur protein dimer binding per double stranded DNAA ligament is shown. Dimer binding was significantly increased at a fur protein concentration of 200 nanomolar, suggesting a high level of non-specific all ligament interactions or a different mechanism for fur protein binding, such as aggregation of multiple dimers.
While attempting this procedure, it is important to remember to start with clean uniform substrates that have the appropriate oxide thickness and to keep the samples clean by carefully handling them with tweezers. It is also crucial to use reagents that have been properly stored and are known to be functional. After watching this video, you should have a good understanding of how to measure biomolecular actions in a label free format through the preparation of functional prob arrays on a silicon dioxide surface, as well as data acquisition and analysis using the interferometric reflectance imaging sensor or iris.
