Chemistry: analytical and immunological testing – Optical result – Including reagent preparation
Reexamination Certificate
2000-10-06
2003-02-04
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
Including reagent preparation
C436S056000, C436S073000, C436S080000, C436S525000, C422S082050, C422S082090, C356S301000
Reexamination Certificate
active
06514767
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to surface enhanced spectroscopy-active composite nanoparticles, methods of manufacture of the particles, and uses of the particles (including their use as molecular or cellular optical tags). More particularly, it is directed to the area of submicron-sized tags or labels that can be covalently or non-covalently affixed to entities of interest for the purpose of quantitation, location, identification, and/or tracking.
BACKGROUND OF THE INVENTION
When light is directed onto a molecule, the vast majority of the incident photons are elastically scattered without a change in frequency. This is termed Rayleigh scattering. However, the energy of some of the incident photons (approximately 1 in every 10
7
incident photons) is coupled into distinct vibrational modes of the molecule's bonds. Such coupling causes some of the incident light to be inelastically scattered by the molecule with a range of frequencies that differ from the range of the incident light. This is termed the Raman effect. By plotting the frequency of such inelastically scattered light against its intensity, the unique Raman spectrum of the molecule under observation is obtained. Analysis of the Raman spectrum of an unknown sample can yield information about the sample's molecular composition.
The incident illumination for Raman spectroscopy, usually provided by a laser, can be concentrated to a small spot if the spectroscope is built with the configuration of a microscope. Since the Raman signal scales linearly with laser power, light intensity at the sample can be very high in order to optimize sensitivity of the instrument. Moreover, because the Raman response of a molecule occurs essentially instantaneously (without any long-lived highly energetic intermediate states), photobleaching of the Raman-active molecule—even by this high intensity light—is impossible. This places Raman spectroscopy in stark contrast to fluorescence spectroscopy, where photobleaching dramatically limits many applications.
The Raman effect can be significantly enhanced by bringing the Raman-active molecule(s) close (<50 Å) to a structured metal surface; this field decays exponentially away from the surface. Bringing molecules in close proximity to metal surfaces is typically achieved through adsorption of the Raman-active molecule onto suitably roughened gold, silver or copper or other free electron metals. Surface-enhancement of the Raman activity is observed with metal colloidal particles, metal films on dielectric substrates, and with metal particle arrays. The mechanism by which this surface-enhanced Raman scattering (SERS) occurs is understood, and is thought to result from a combination of (i) surface plasmon resonances in the metal that enhance the local intensity of the light, and; (ii) formation and subsequent transitions of charge-transfer complexes between the metal surface and the Raman-active molecule.
SERS allows detection of molecules attached to the surface of a single gold or silver nanoparticle. A Raman enhancing metal that has associated or bound to it a Raman-active molecule(s) is referred to as a SERS-active nanoparticle. Such SERS-active nanoparticles can have utility as optical tags. For example, SERS-active nanoparticles can be used in immunoassays when conjugated to an antibody against a target molecule of interest. If the target of interest is immobilized on a solid support, then the interaction between a single target molecule and a single nanoparticle-bound antibody could be detected by searching for the Raman-active molecule's unique Raman spectrum. Furthermore, because a single Raman spectrum (from 100 to 3500 cm
−1
) can detect many different Raman-active molecules, SERS-active nanoparticles may be used in multiplexed assay formats.
SERS-active nanoparticles offer the potential for unprecedented sensitivity, stability, and multiplexing functionality, when used as optical tags in chemical assays. However, SERS-active nanoparticles made from metals present formidable practical problems when used in such assays. Metal nanoparticles are exceedingly sensitive to aggregation in aqueous solution; once aggregated, it is not possible to re-disperse them. In addition, the chemical compositions of some Raman-active molecules are incompatible with the chemistries used to attach other molecules (such as proteins) to metal nanoparticles. This restricts the choices of Raman-active molecules, attachment chemistries, and other molecules to be attached to the metal nanoparticle.
The most significant problem with the use of metal nanoparticles as Raman tags is the similarity of the Raman spectra of molecules to be coupled to the nanoparticles. For example, in a multiplexed sandwich immunoassay, the Raman spectra of the secondary antibodies to which the nanoparticles are attached would be highly similar, and thus impossible to deconvolute. Moreover, the parts of the secondary antibodies that are different, i.e., the antigen-binding domains, are typically too far away from the metal surface to be significantly enhanced.
The prior art teaches that molecules themselves can be used as Raman tags, provided that their Raman scattering cross section is sufficiently large. Thus, direct attachment of dyes, for example, to antibodies, allows them to be used as tags for immunoassays. This approach, however, suffers from extremely significant limitations: the molecular structures/features that give rise to intense Raman spectra (e.g. polarizability, aromaticity, conjugation, heteroatoms, and most significantly, significant absorption cross section) also give rise to complex Raman spectra. The use of molecular Raman tags requires very high extinctions in the visible region of the spectrum to access resonance Raman scattering, which increases the Raman signal by up to three orders of magnitude. There is a fundamental physical incompatibility between molecules that absorb visible light well and those that exhibit simple Raman spectra. Thus, the Raman spectra of the dyes described above are exceedingly complex, and it has not been possible to multiplex these assays.
A second fundamental problem with Raman-based tags is the weakness of the Raman signal; it is not possible to detect single molecules (or even thousands of molecules) by Raman without using surface enhancement. Ideally, one would like a tag that exhibits the enhancement factors associated with SERS and the ability to attach such a tag to a freely diffusing species (which would clearly not be possible with macroscopic SERS-active surfaces).
It is an object of this invention to provide a solution to the abovementioned problems encountered when using Raman scattering entities as optically-addressable labels or tags, especially in chemical or biomolecular assays. It is a further object of the invention to provide a panel of at least 20 different SERS-active nanoparticles for use as “cleaveless” optical tags in bead-based combinatorial chemical syntheses. It is a further object of this invention to describe an optical detection system for multiplexed assays.
The present invention is directed to surface enhanced spectroscopy-active composite nanoparticles, including SERS-active composite nanoparticles (SACNs). Also included within the scope of this invention are methods of manufacture of the particles, and uses of the particles (including their use as molecular or cellular optical tags). The submicron-sized-tags or labels of the invention can be covalently or non-covalently affixed to entities of interest(that may range in size from molecules to macroscopic objects) for the purpose of quantitation, location, identification, and/or tracking.
SUMMARY OF THE INVENTION
The present invention overcomes the problems encountered when using a spectroscopy-active species, such as a Raman scattering species, as an optical tag. The invention provides novel SES-active composite nanoparticles, including SERS-active composite nanoparticles (SACNs). Such nanoparticles each comprise a SES-active metal nanoparticle, a submono
Gakh Yelena
SurroMed, Inc.
Swanson & Bratschun L.L.C.
Warden Jill
LandOfFree
Surface enhanced spectroscopy-active composite nanoparticles does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Surface enhanced spectroscopy-active composite nanoparticles, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Surface enhanced spectroscopy-active composite nanoparticles will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3128756