The present invention is directed toward a SERS nanotag and more particularly toward a SERS nanotag having a polymer coating.
SERS nanotags are glass coated metal nanoparticles that produce a strong Raman scattering signal when excited by visible and near infrared light. SERS nanotags may be used to perform in vivo assays. Native nanotags, however, appear to the body as foreign objects and so will usually be cleared by the body quickly.
The present invention is directed toward overcoming one or more of the problems discussed above.
I. Introduction
SERS nanotags are glass coated metal nanoparticles that produce a strong Raman scattering signature when excited by visible and near infrared light. They may be used to perform In-Vivo assays where specific physiological regions, cells, tumour, tissues etc. are targeted by the SERS nanotags as a diagnostic label similar to a fluorophore. Native particles will appear to the body as foreign bodies and so will usually be cleared by the body quickly. Coating in vivo diagnostic particles in polymers can reduce the rate at which particles are cleared by the body.
SERS nanotags are glass coated and so can be subsequently coated by a variety of different molecules, using a variety of different attachment methods. Polymers are coated on particles to increase their retention time the body. Typical polymers used are PEGS (polyethylene glycol), Dextrans etc. PEGS used typically need to be greater than 5000 Da. Particles can be further modified by attaching proteins or antibodies specific for selected physiological regions. Indeed the flexibility of glass attachment chemistry means that a combination of polymers and proteins can be employed which will allow the user to optimize site specificity and retention time. Other particles, e.g Quantum Dots, cannot be as easily coated with a variety of polymers. Therefore the SERS nanotags ability to be retained by the circulation system should be greater than that of Quantum Dots and other particles.
II. SERS Nanotags
One embodiment of the present invention includes the use of encapsulated surface enhanced Raman scattering (SERS) tags. These nanoparticles, referred to as SERS nanotags, include a metal nanoparticle, which metal is Raman enhancing; a Raman-active molecule (sometimes referred to as a SERS tag or reporter molecule) attached to, or associated with the surface of the nanoparticle; and an encapsulant, usually SiO2 (glass). The encapsulant surrounds both the metal nanoparticle and the Raman-active molecule. A particle prepared in this fashion has a measurable SERS spectrum. Although the invention is described in terms of SERS nanotags prepared from single nanoparticles, it is to be understood that nanoparticle core clusters or aggregates may be used in the preparation of SERS nanotags. Methods for the preparation of clusters of aggregates of metal colloids are known to those skilled in the art. The use of sandwich-type particles is described in U.S. Pat. No. 6,861,263, which patent is incorporated herein by reference.
SERS data may be obtained from the tags by illuminating the SERS nanotags with a suitable excitation wavelength. In the case of some reporter molecules excitation wavelengths are in the range of about 600-1000 nm. In some embodiments, the excitation wavelengths are 632.8, 785, or 980 nm. Examples of reporter molecules include 4-mercaptopyridine (4-MP); trans-4,4′ bis(pyridyl)ethylene (BPE); quinolinethiol; 4,4′-dipyridyl, 1,4-phenyldiisocyanide; mercaptobenzamidazole; 4-cyanopyridine; 1′,3,3,3′,3′-hexamethylindotricarbocyanine iodide; 3,3′-diethyltiatricarbocyanine; malachite green isothiocyanate; bis-(pyridyl)acetylenes; Bodipy, and isotopes thereof, including, for example, deuterated BPE, deuterated 4,4′-dipyridyl, and deuterated bis-(pyridyl)acetylenes; as well as pyridine, pyridine-d5 (deuterated pyridine), and pyridine-15N. A suitable excitation wavelength is one at which the background noise component, generated by fluorescence from other fuel components is low enough to obtain a detectable SERS signal.
The SERS nanotags may comprise any nanoparticle core known in the art to be Raman-enhancing. As used herein, the term “nanoparticle”, “nanostructure”, “nanocrystal”, “nanotag,” and “nanocomponent” are used interchangeably to refer to a particle, generally a metallic particle, having one dimension in the range of about 1 nm to about 1000 nm. In some embodiments, the metal nanoparticle core is a spherical or nearly spherical particle of 20-200 nm in diameter. In some embodiments the range is about 20 nm to about 50 nm, in some embodiments in the range of about 30 nm to about 100 nm. The tags may be polydisperse. That is, a group of tags may comprise tags with these ranges of diameters, but each tag need not have the same diameter.
Nanoparticles may be isotropic or anisotropic. Anisotropic nanoparticles may have a length and a width. In some embodiments, the length of an anisotropic nanoparticle is the dimension parallel to the aperture in which the nanoparticle was produced. In the case of anisotropic nanoparticles, in some embodiments, the nanoparticle has a diameter (width) of 350 nm or less. In other embodiments, the nanoparticle has a diameter of 250 nm or less and in some embodiments, a diameter of 100 nm or less. In some embodiments, the width is between 15 nm to 300 nm. In some embodiments, the nanoparticle has a length of about 10-350 nm.
Nanoparticles include colloidal metal, hollow or filled nanobars, magnetic, paramagnetic, conductive or insulating nanoparticles, synthetic particles, hydrogels (colloids or bars), and the like. The nanoparticles used in the present invention can exist as single nanoparticles, or as clusters or aggregates of the nanoparticles. Clusters or aggregates may be formed by the addition of aggregating agents to the SERS nanotags.
It will also be appreciated by one of ordinary skill in the art that nanoparticles can exist in a variety of shapes, including but not limited to spheroids, rods, disks, pyramids, cubes, cylinders, nanohelixes, nanosprings, nanorings, rod-shaped nanoparticles, arrow-shaped nanoparticles, teardrop-shaped nanoparticles, tetrapod-shaped nanoparticles, prism-shaped nanoparticles, and a plurality of other geometric and non-geometric shapes. Another class of nanoparticles that has been described include those with internal surface area. These include hollow particles and porous or semi-porous particles. Moreover, it is understood that methods to prepare particles of these shapes, and in certain cases to prepare SERS-active particles of these shapes, have been described in the literature. While it is recognized that particle shape and aspect ratio can affect the physical, optical, and electronic characteristics of nanoparticles, the specific shape, aspect ratio, or presence/absence of internal surface area does not bear on the qualification of a particle as a nanoparticle.
Various systems can be used for detection of SERS nanotags. A number of commercially available instruments may be used. For example, Raman Systems Inc., Enwave Optronics, Inc., Kaiser Optical Systems, Inc., InPhotonics, Inc., J-Y Horiba, Renishaw, Bruker Optics, Thermo Electron, Avalon, GE Ion Track, Delta Nu, Concurrent Analytical, Raman Systems, Inphotonics, ChemImage, Jasco, Lambda Systems, SpectraCode, Savante, Real-Time Analyzers, Veeco, Witec, and other companies provide Raman spectrometers suitable for use in the present invention.
III. Polymer Coated SERS Nanotags
The glass coated SERS nanotags described above can be derivatized with polymers using a variety of methods.
The native glass coat serves at least 2 purposes:
These surfaces are amenable to the development of robust and controllable methods for bioconjugation. Indeed, the surface silanol groups can be easily derivatized with commercially available mercapto-, carboxy-, amino-, aldehydo- and epoxy-silane reagents.
The introduction of the functional groups has been done by 2 alternate routes:
These functionalization routes provide the flexibility to conjugate practically any type of molecule. This method takes advantage of the large library of functional PEGs provided by Nektar (form. Shearwater) to generate many PEGylated tags. (See
PEG may thus provide biocompatibility and extended in-vivo lifetimes of the SERS tags.
To achieve a similar extended bioavailability the tags can alternatively be coated with other molecules such as with proteins, DNA, RNA, synthetic Polyaminoacids (Polylysine, Polyglutamic acid), Polyethylene glycols, block copolymer dendrimers, polyamides, polyethylenimines, polyacrylates and other natural polymers such as Dextrans and other natural carbohydrate based polymers
The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
Functionalization of Glass coated SERS tags using the 2 Glass-layer approach:
Materials & Reagents
Amino-Tags: APTMS derivatization of Glass coated SERS tags
Epoxy-Tags: GPTMS derivatization of Glass coated SERS tags
Thiolated-Tags: MPTMS derivatization of Glass coated SERS tags (this is the protocol for conventional tag preparation)
Carboxy-Tags: CEST derivatization of Glass coated SERS tags
General Direct derivatization method for functionalization of Glass coated SERS tags
Derivatization of Functional Glass-coated SERS Tags with PEG derivatives
Amino-Tag derivatization with mPEG-SPA, Fluorescein-PEG-NHS & Succinic Anhydride
Materials & Reagents
Amine derivatization with Succinic Anhydride
Amine derivatization with mPEG-SPA
Amine derivatization with Fluorescein-PEG-NHS
Epoxy-Tag derivatization with mPEG-NH2 & NH2-PEG-Carboxylate
Materials & Reagents
Epoxide reaction with mPEG-NH2
Epoxide Reaction with NH2-PEG-Carboxylate
Thiolated-Tag derivatization with Maleimido-mPEGs
Materials & Reagents
Thiol reaction with Maleimido-mPEG-5,000
Thiol reaction with Maleimido-mPEG-20,000
While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.
This application claims priority from U.S. Provisional Application Ser. No. 60/758,873, filed on Jan. 13, 2006, entitled “Polymer Coated SERS Nanotag”, the contents of which are incorporated herein in their entirety.
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