The present invention relates to surface plasmon resonance detection and measurement. It relates especially to the application of this technique to perform biological assay measurements reliably on a high volume basis.
1. Field of the Invention
Surface plasmon resonance (SPR) is an established technique for sensing and measuring refractive index changes in materials and especially for the label-less detection of biological and biochemical assays. Surface plasmon resonance biosensors use surface plasma waves to probe biomolecular interactions occurring at the surface of a sensor; see e.g. U.S. Pat. No. 4,844,613 (Batchelder et al.) and 4,997,271 (Finlen et al.).
Measuring instruments incorporating SPR detectors having a variety of optical geometries have been used to obtain SPR signals. One widely used configuration known from the former patent above is shown diagrammatically in FIG. 1. There, a light source 2 directs diverging monochromatic light rays 3, i.e. the interrogation beam, spanning a range of angles at the entrance surface 4a of a high index triangular glass prism 4. The rays are refracted upon passing into the prism where they impinge upon a prism surface 4b, i.e. a sensing surface, coated with a thin layer 5 of metal, e.g. gold. The fan of rays is reflected from that surface and exits the prism 4 from an exit surface 4c. The reflected light rays 6 impinges upon an imaging detector array 7 that produces an output signal which, when processed properly, may control a display device to produce an image 8.
As seen from FIG. 1, the image 8 has a substantially uniform intensity except at a small band or feature 8a projecting the angle attenuated by the SPR effect where the image is significantly less bright. An example of a reflection verses angle function of the image 8 produced by the FIG. 1 instrument as illustrated in FIG. 2. In angular space, the relatively dark attenuation band or feature 8a is typically less than 1° wide and the instrument detects and measures the location of that feature. The angle at which maximum attenuation of a particular wavelength of the incident light occurs is dependent upon the index of refraction of a layer 9 of a material deposited on top of metal layer 5. Thus the movement of the dark feature 8a in the overall image 8 reflects refractive index changes in the material layer 9 on the metal layer 5.
While the SPR detector illustrated in FIG. 1 has the metal coating 5 applied directly to the prism, most detectors of this type used to perform assays, e.g. for biological screening, have the metal layer on a separate flat optical element or plate which is optically coupled to the prism. Also other prism shapes are possible.
FIG. 3 shows a SPR detector known from the latter patent above, wherein the metal layer 10 is deposited on a flat glass plate 12 which forms the bottom wall of a flow cell 14 defining a cavity 14a containing a sample 16 which contacts the metal layer 10 within the cavity. The flow cell 14 including the coated plate 12 may constitute a disposable unit 17 which is removably coupled by an index matching liquid 18 such as oil, to the flat surface 20a of a glass prism 20, in this example a hemispherical or semicylindrical prism, which is usually a fixed part of the measuring instrument. Interrogating light rays 22, in this case a converging beam, enter one side of prism 20 and are brought to a focus at a point P located at the metallized surface of plate 12 near the center of curvature of prism 20. Light which is internally reflected at point P passes out through the opposite side of the prism as measuring rays 24 which impinge on a detector array 26. As in the FIG. 1 instrument, detector 26 produces an image having a dark feature whose position in the image reflects the index of refraction of sample 16 in the flow cell 14.
SPR assay implementations using flow cells such as the one in FIG. 3 have a relatively low throughput because it takes time to pump enough of a particular sample into the flow cell cavity to clear material from a previous measurement in that same cell and to stabilize a new concentration of material for the new measurement. Also as noted above, index matching liquid 18 must be present between the flow cell unit 17 and the surface 20a of prism 20. This fluid must be applied consistently with no bubbles before coupling the flow cell unit to the prism. Moreover, flow cell unit separation from the prism after measurement is difficult because of the need to overcome surface tension between the opposing surfaces of the unit 17 and the prism. Still further, the liquid 18 must be cleaned from the prism and from the flow cell unit after each assay to avoid the buildup of contaminants which could degrade the next measurement using that same unit. Such required maintenance steps slow down the SPR measurements and reduce the throughput of the measuring instrument as a whole.
Because of the inconvenience of having to repeatedly couple and decouple the flow cell unit to the fixed prism using the index matching liquid and also because of the expense of the flow cell unit per se, these units are often reused for multiple assays or tests. Researchers have validated processes that accommodate such reuse. However, concerns about potential cross-contamination between samples have to be addressed following complex disassociation and washing protocols which are time consuming and reduce the throughput of the measuring instrument as a whole. Moreover, such re-use of the flow cell units bucks the overall trend towards the use of disposable labwear in life science research.
In biological screening, large numbers of assays have to be performed in parallel. Likewise in many life science research or drug discovery settings, multiple samples need to be measured in a single session. For these reasons, molded microplates defining a multiplicity of sample wells are very commonly used in these fields for performing assays with signal-producing label chemistries such as fluorescence, luminescence or radioactivity. Microplates having 96, 384 and 1586 wells are made to standardized dimensions prescribed by the Society for Biomolecular Screening (SBS). Considerable instrumentation, automation and infrastructure have been developed over the years at life science research and drug discovery institutions to accommodate assays using these microplates. However, until now no one has thought to incorporate SPR detection features into a microplate-type structure because of the seemingly insurmountable obstacle to interrogating and measuring samples in the densely packed wells of the microplate.
Therefore, it would be desirable if there existed SPR detection and measuring apparatus that combines the simplicity and low cost of a fixed-prism measuring instrument with the throughput and ease of handling of a standard-format microplate.