Surface plasmon resonance imaging (SPRi) brings label-free detection of molecular interactions to microarray analysis. The arrayed molecules (probes) may be proteins, peptides, nucleic acids, sugars, membranes, or any other molecule of interest.
The analyte, which may likewise be any type of biological sample, is flowed over the entire array so that all probes are exposed to exactly the same solution and all measurements are collected simultaneously. In this way, the array format increases throughput and simultaneously measures all the probes in the same solution and eliminates channel-to-channel variation.
Label-free systems are widely appreciated in proteomics because there is no need to modify proteins chemically with fluorescent or other tags that might compromise protein function. Less appreciated is the fact that SPRi systems provide real-time data, which enables the user to monitor the progress of binding without disturbing molecular interactions.
With the SPRimager®II from GWC Technologies, real-time data is presented as charts that quantify molecular binding to the array and also as array images for evaluation by that most powerful of scientific instruments, the human eye. Difference images obtained by digital subtraction of a reference image from a postbinding image allow for visual confirmation of binding: where binding occurs, reflectivity increases and spots become brighter.
In order to maximize versatility, two types of array substrates (chips) are offered for this platform:
• SpotReady™ chips have 16 or 25 gold spots on a hydrophobic surface. Arrays are made by spotting ~0.5 µL of probe solution per spot using a regular micropipette. This format requires no spotting robot and is ideal for arrays where no more than 25 probes are needed, where probe solution is scarce, or for rapid methods development.
• The SPRchip™ has a uniform gold surface suitable for robotic spotting of higher-density arrays in the user’s preferred layout.
In a typical experiment, arrays are fabricated at the bench by first activating the gold surface with a functional group for attachment of probe molecules. For example, proteins can be covalently attached to the chip via lysine amine groups by any of the popular methods; biotinylated probes (whether antibodies, peptides, oligonucleotides, or proteins) can be captured on a streptavidin-modified surface; and oriented arrays of mAbs can be prepared on goat-antimouse (Fc) surfaces.
After fabrication, the array is assembled into the SPRimager II flow cell for analysis. Analyte is pumped over the array and binding on each spot is monitored and plotted as changes in reflectivity vs. time. Both reflectivity increases and decreases are readily measured, which allows for monitoring of both association and dissociation.
SPRi has been used for diverse applications such as characterizing protein-protein and antibody–antigen interactions; determining affinities, on-rates and off-rates for molecular interactions; monitoring protein binding to aptamer arrays; analyzing transcription factor binding to DNA arrays; profiling drug-absorption characteristics on cell membrane arrays; and profiling cell-surface receptors on ligand arrays. In this article, the utility of the SPRimager II system is illustrated with three examples.