Chemistry: analytical and immunological testing – Optical result
Reexamination Certificate
2000-11-13
2003-06-17
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
C436S525000, C356S445000
Reexamination Certificate
active
06579721
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to surface plasmon resonance sensors. In particular, the present invention relates to instruments, methods and reagents for amplifying the surface plasmon resonance response and increasing its sensitivity, flexibility, throughput, specificity, and scope.
BACKGROUND OF THE INVENTION
Surface plasmon resonance (SPR) is a general spectroscopic method for sensing refractive index changes near the surface of a metal film. Its sensitivity to these changes provides a versatile platform for the observation and quantitation of chemical reactions at the metal/solution interface, provided the chemistry is well-designed. The generality of the technique has led to its application to a variety of chemical systems, including biosensing (where specifically designed commercial instrumentation is available).
SPR allows detection of small changes in refractive index that result from interactions between surface-confined biomolecules and solution-borne species. For example, immobilization of a protein to the sensor surface allows for detection of protein binding events manifested by a change in refractive index and hence a change in the angle-dependent reflectance of the metal film. This type of SPR sensing is typically carried out on commercial instruments that use a carboxylated dextran gel on a Au film as the sensor surface, where the gel acts as a host for the surface-confined binding partner. However, SPR has been applied in a number of other formats, including imaging SPR where a large number of chemistries can be rapidly interrogated simultaneously. Likewise, a variety of other surface chemistries have been used as scaffolds for measurements of biomolecular interactions.
SPR relies on the optical excitation of surface modes (plasmons) in a free electron metal (e.g., a 50 nm thick film of Au, Ag, Al, or Cu anchored to a glass substrate by a thin adhesion layer of Ti, Cr, or mercaptosilane). Back-side, p-polarized illumination of a prism-coupled film at some angle greater than the critical angle for total internal reflection results in plasmon excitation at the metal-solution interface. Plasmon excitation is observed as an increase in optical absorbance (decrease in reflectance) at an optimal coupling angle. This, in turn, results in a minimum in the SPR profile (a plot of reflectance versus angle), which is referred to as the plasmon angle (&thgr;
p
). Sensing via SPR is possible due to the sensitivity of &thgr;
p
to changes in the index of refraction near the metal surface. Adsorption, desorption, and molecule-molecule interactions that occur at the metal-solution interface result in such changes, thereby inducing a shift in plasmon angle. These changes can be monitored in real-time, making SPR suitable for dynamic sensing.
Perhaps the most widely studied subset of chemistries using SPR is protein-protein interactions, where binding event signal transduction is difficult or impossible to accomplish by traditional optical spectroscopies. In order to decrease nonspecific binding and to increase surface loading of biomolecules, SPR experiments conducted on commercial instrumentation commonly use an extended coupling matrix in conjunction with the sensing surface. Such measurements typically begin with one protein immobilized on proprietary substrates comprising a carboxylated dextran (or “extended coupling”) matrix layered on top of a thin evaporated Au film (i.e., between the film and the sample). The result is an extended three-dimensional array of molecules extending some 200 nm away from the surface of the Au film. Protein binding events leading to small changes in the refractive index of the dextran layer are detected via correspondingly small changes in the angle-dependent attenuated total reflectance. Despite the signal amplification afforded by the dextran matrix, the detection of small (<1000 MW) molecules is still a non-trivial task for commercial instrumentation. In some cases even the detection of species in the 2,000-10,000 MW range can prove challenging.
Use of coupling matrices is associated with a number of additional drawbacks, including the possibility of nonspecific interactions with biomolecules that dominate the signal, and the exclusion of large proteins. Furthermore, improper orientation of biomolecules within the matrix often leads to low biomolecular activity, especially with proteins. Because mass transport to molecules immobilized in the matrix is commonly pH dependent, separate steps are required to optimize diffusion of reagents into the matrix during the assay. These shortcomings can create significant difficulties in assay design. It would be desirable to avoid these problems by using planar SPR substrates (i.e., without an overlying matrix) such as Au films modified with a monolayer of a bifunctional organic crosslinker. However, SPR reflectivity changes within these more uniform substrates are often too small to be measurable in any practical assay.
The applicability and usefulness of SPR could be greatly expanded if ligand binding events resulted in more pronounced changes in refractive index and, hence, more pronounced shifts in plasmon angle. Such increased sensitivity could make the technique broadly applicable to high-throughput screening of low molecular-weight drug candidates.
It is an object of the present invention to provide instruments, methods and reagents for the amplification of SPR reflectivity changes in chemical assays, especially in biomolecular recognition assays on planar SPR substrates coated with a monolayer or submonolayer of capture reagents (e.g., antibodies). It is also an object of the invention to provide methods and reagents for ultra-sensitive, non-PCR-based DNA detection assays. It is moreover an object of this invention to describe an SPR-based method for measuring distances between surface-confined objects on the scale from 1 to 500 nm, with 3-Angstrom resolution.
An additional object of this invention is to enable high throughput screening reactions (both primary and secondary) by amplified imaging SPR.
A further object of the invention is to provide a “wet chemistry” method for the synthesis of Au films for SPR (or other surface spectroscopies) as a replacement for (or alternative to) cumbersome evaporative methods used in the art.
Finally, it is an object of the invention to provide an imaging SPR instrument capable of depicting spatial differences in film reflectance at fixed angles of incidence, which spatial differences are induced by differential indices of refraction or film thickness (i.e., differential chemical modification). The imaging SPR instrument provided by the invention allows SPR to be used in multiplexed biological “chip” assay formats. For example, the imaging SPR instrument would be integral in the simultaneous detection of multiple target analytes using a solid support to which ligands for the different target analytes are attached at specific locations.
The instrumentation, methods, and reagents of the present invention enable chemical assays (including multiplexed biosensing assays) of unprecedented sensitivity and selectivity.
SUMMARY OF THE INVENTION
The present invention provides instrumentation, methods, and reagents for the amplification of SPR reflectivity changes. In one series of embodiments, colloidal-metal nanoparticles are used as optical tags for SPR-based sensing assays. These embodiments rely on the observation that the SPR response of a metal film changes dramatically upon localization of such colloidal-metal nanoparticles to the film surface. The dramatic change in the SPR response of metal films that occurs upon adsorption of colloidal metal nanoparticles can be exploited in any assay that depends on the occurrence of a molecular recognition event (e.g., the binding of antigen to an antibody, the binding of a ligand to its receptor, or the hybridization of complementary nucleic acid molecules). In one of the many possible implementations, one of the molecules that participates in a molecular recognition event is immobili
Goodrich Glenn
He Lin
Holliway William D.
Lyon L. Andrew
Musick Michael D.
Snay Jeffrey
SurroMed, Inc.
Swanson & Bratschun LLC
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