1Department of Electrical and Computer Engineering, Boston University, 2Department of Biomedical Engineering, Boston University, 3Center for Advanced Genomics Technology, Boston University, 4Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 5Department of Microbiology, Boston University School of Medicine, 6CNR (National Research Council), Istituto di Chimica del Riconoscimento Molecolare
Lopez, C. A., Daaboul, G. G., Ahn, S., Reddington, A. P., Monroe, M. R., Zhang, X., et al. Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS). J. Vis. Exp. (51), e2694, doi:10.3791/2694 (2011).
The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.
1. Substrate Preparation
2. Preparation of Probe Array: Antibodies, antigens, ss/dsDNA, RNA, etc.
3. Data Acquisition and Incubation Procedure: Endpoint format
4. Data Analysis
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).
The IRIS platform is a simple and rapid system to use for collecting high-throughput binding data in a microarray format. By covalently immobilizing different functional protein or DNA probes on a surface, target antigens/biomarkers/etc. can be captured from solution, as in an immunoassay. Measurement of these interactions across probe conditions in an endpoint or real-time format with the IRIS system allows for highly sensitive and quantitative information to be collected for each interaction. The representative experiment detailed here is just one example of the types of interactions that can be studied with this technique. Detection of transcription factors, viral antigens, and whole viruses has been demonstrated previously and represents just a few examples of the types of analyses that the IRIS can easily handle.
No conflicts of interest declared.
The authors wish to thank Zoiray Technologies Inc., a co-sponsor and commercialization partner, for its support.
|Silicon wafers with 500 nm of thermally-grown silicon dioxide||Silicon Valley Microelectronics|
|Ammonium sulfate powder||Sigma-Aldrich||A5132|
|Phosphate buffered saline (PBS) 10X ready concentrate||Fisher Scientific||BP665-1|
|Antibody to human serum albumin (Anti-HSA)||Sigma-Aldrich||A0433|
|Human serum albumin (HSA)||Sigma-Aldrich||A9511|
|Argon or nitrogen gas (ultra-high purity)||Airgas||AR UHP300 or NI UHP300|
|Petri dishes, 60 mm x 15 mm||Fisher Scientific||0875713A|
|SciencewareÂ® vacuum desiccator||Sigma-Aldrich||Z119016|
|Microcentrifuge tubes||Fisher Scientific||05-408-120|
|96-well plates, low cell binding||Nalge Nunc international||145399|
|BioOdyssey Calligrapher MiniArrayer||Bio-Rad|
|Hydroxylamine||Thermo Fisher Scientific, Inc.||26103|
|Multi-purpose rotator, model # 2314||Thermo Fisher Scientific, Inc.||2314Q|
|Retiga 2000R camera||QImaging||01-RET-2000R-F-M-12|
|AcuLED illumination source||PerkinElmer, Inc.|
|Optical components (lenses, objectives, apertures, rails, posts, etc.)||Thorlabs Inc.|