March 1, 2008 (Vol. 28, No. 5)
Voula Kodoyianni, Ph.D.
SPRi Does Not Need to Be Limited to Purified Analytes, Lysates, or Extracts
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.
Array Quality and Function Analysis
A 400-spot protein array was fabricated on an N-hydroxysuccinimide-activated (NHS) SPRchip using the GeSiM® Nanoplotter with nanotip (HTS Resources) for analysis on the SPRimager II (Figure 1). The three proteins (protein G, b2 microglobulin, and streptavidin) were spotted at the same concentration (0.5 mg/mL).
Analysis of the array image prior to exposure to analyte reveals that there is little interspot variation within protein blocks but that different amounts were actually immobilized for each protein (Figure 1, top panel). This difference in mass cannot be explained by the difference in molecular weights alone and appears to be highly protein dependent. This analysis illustrates the built-in quality control of SPRi and shows that the SPRimager II can provide critical information on array quality prior to experimental analysis.
To analyze protein function, the array was then exposed to three different analyte solutions sequentially (Figure 1). Each analyte bound specifically to its probe demonstrating protein functionality was preserved. Replicate probes for each protein bound reproducible quantities of analyte (Table). Difference images obtained after exposure to each analyte was complete (Figure 1, bottom panel) show specific binding where expected.
Knowledge of the epitope to which an antibody binds is important for many antibody application areas. In this example, a simple method is presented for identifying suitable antibody pairs for diagnostic assays where two antibodies—capture and detection—are required that recognize the same antigen via distinct epitopes.
Here, antibodies raised to the same RNA polymerase antigen were arrayed on a SpotReady chip using a goat-antimouse affinity surface for immobilization. The array was then exposed first to antigen, then sequentially to each of the same three antibodies on the array, binding events are monitored on the SPRimager II.
As illustrated (Figure 2, left), antigen should, of course, bind to all antibodies, but subsequent exposure to each antibody will result in binding only if the epitope on the antigen is available, i.e., not bound to the immobilized antibody.
Figure 2 (right) shows the results. Each antibody bound to the antigen captured by the other two antibodies, but not to itself, which indicates that all three antibodies recognize distinct epitopes on this antigen. Denser arrays could readily be used to perform the same type of analysis on many antibodies simultaneously.
No dextran gels or other matrices are used on SpotReady or SPRchip substrates, in order to provide optimal access of analytes to the probes on the arrays. While this has obvious benefits for accurate kinetics analysis, it is also convenient for analyzing large analytes, in this example, whole cells.
Here, a protein ligand array was fabricated on a SpotReady chip using covalent attachment to an NHS surface. Three proteins were immobilized, basic fibroblast growth factor (bFGF), cytochrome c, and insulin. BHK21 cells in buffer were then flowed over the array to determine whether the cells would specifically bind to ligands for which they have cell-surface receptors.
As Figure 3 shows, the cells bound strongly to bFGF, a ligand for which receptors are abundant on the cell surface. The cells also bound moderately to insulin, implying the presence of insulin receptors on these cells, but they did not bind significantly to the cytochrome c control. These results show that ligand arrays can be used on the SPRimager II platform to characterize cell-surface receptors simply by flowing whole cells over the array. An array with many ligands could provide information on the presence of multiple receptors in a single experiment.
More generally, this experiment shows that SPRi analysis need not be limited to purified analytes, lysates, or extracts but may also be applied to analyzing massive targets. In addition to mammalian cell-receptor analysis, this approach could be used to characterize bacterial and viral pathogens in food and monitor virion assembly on nucleic acid templates.