Patent Application
20070099238
Fluorescent dyes are used for fluorescent tagging in bioimaging and biosensor applications. Photon absorption in dyes produces a fluorescence band that is typically red-shifted from the incident photon frequency. The difference in energy between the absorbed photon and the emitted photon corresponds to the energy loss due to nonradiative processes. Metal coated spheres containing dyes have been proposed for field enhancement in Raman spectroscopy by C. Oubre and P. Nordlander in Journal of Physical Chemistry B, 108, 17740 (2004).
In accordance with the invention, the efficiency of fluorescent dyes or quantum dots using metal coated dielectric spheres or other metal coated dielectric shapes may be improved. Fluorescent dyes or quantum dots may be embedded in a dielectric volume of appropriate dimensions where typically half the surface of the dielectric volume is covered by a metal coating allow for increased absorption and emission efficiencies. Alternatively, fluorescent dyes or quantum dots may be attached to metal coated dielectric shapes using the appropriate chemistries.
a-b steps for a photochemical analytic sequence in accordance with the invention.
In
Metal coating 145 is a silver coating for curves 210, 215, 220 and 225. Metal coating 145 functions as a reflector and creates a resonant cavity so that the TRP ratio is a maximum at resonance. Increasing the thickness of metal coating improves the reflective properties to increase TRP ratio while increasing the resonance frequency as seen by curves 210 and 220. However, for thick metallic coatings 145 thicker than about 20 nm on fully metal coated dielectric nanospheres 150, the losses in metal coating 145 typically become dominant and TRP ratio decreases. Although the above curves 210, 215, 220 and 225 were calculated for spheres, similar enhancements of TRP ratio occur for other geometrical shapes such as, for example, partially or fully metal coated cubes or cylinders.
In
Calculations where both the diameter of nanosphere 150 and the thickness of metal coating 145 scale accordingly indicate that the resonance frequency stays about the same. This result is expected in view of the behavior shown in
a-4b show the steps for using half metal coated dielectric nanospheres 150 in a photochemical analytical sequence. Because dielectric nanospheres 150 are only half covered with metal coating 145, selective chemistry may be performed on the surface of dielectric nanosphere 150 and on the surface of metal coating 145 in order to orient metal coating 145 to a predetermined orientation to act as a receiving cup for the incoming pump light. The relevant literature provides the specific chemistries that have been developed for attaching bio-molecules to specific surfaces such as Au, Ag, SiO2, polystyrene or latex. If a molecular entity that is sensitive to the specific emission wavelength of dielectric nanaosphere 150 is attached to dielectric nanosphere 150 other functions may be performed. The enhanced TRP ratio provided in accordance with the invention allows the efficient optical excitation of the molecular entity at very low concentrations which has applications in bio-sensing applications, for example.
Initially, functionalized surface 410 is placed in a solution containing half metal coated dielectric nanospheres 150. The half metal coated dielectric nanospheres 150 are prepared with appropriate antibodies 415 attached to metal coating 145. If metal coating 145 is gold, a thiol based chemistry that is complimentary to functionalized surface 410 is typically used. Typically, second set of antibodies 435 may be attached to the exposed dielectric portion of half metal coated dielectric nanospheres 150, second set of antibodies 435 is of relevance to the subsequent chemistry that is to be performed. Half metal coated dielectric nanospheres 150 can then be positioned with metal coating 145 adjacent to the functionalized surface 410 so that the exposed dielectric portion of half metal coated dielectric nanospheres 150 is directed towards pump light 450, attached antibodies 435 dangling radially. The procedure is performed with a variety of properly functionalized half metal coated dielectric nanospheres 150 tagged with appropriate dyes that each in turn will bind specific molecular entities 470 of interest. To perform an assay on a variety of tagged molecular entities 470, dye or quantum dot doped half metal coated dielectric nanospheres 150 are optically pumped at an appropriate wavelength to optically excite the appropriate dye or quantum dot doped half metal coated dielectric nanosphere or nanospheres 150. The wavelength is down shifted and re-emitted efficiently because of enhanced directionality. The efficient absorption of pump light 450 together with the highly directional and efficient re-emission of the light allows detection of molecular entities at very low concentrations. After half metal coated dielectric nanospheres 150 bind to the functionalized surface, half metal coated dielectric nanospheres 150 are typically exposed to light for photochemical analysis.
In addition to the geometry shown in
Dye-doped dielectric nanospheres 150, typically of latex or polystyrene of various sizes are commercially available, for example, from MOLECULAR PROBES CORPORATION. Synthesis of dye-doped dielectric nanoparticles of silica using a micro-emulsion method is described by S. Santra et al in Journal of Biomedical Optics 6, 160-166 (2001) and incorporated herein by reference. The silica nanoparticles can be made in uniform sizes with typical diameters from a few nanometers to a few micrometers with the size distribution controlled to within about two percent.
In accordance with the invention, dye-doped dielectric nanospheres 150 may be replaced with quantum dot doped dielectric nanospheres 150. Unlike dye molecules, quantum dots absorb light at short wavelengths and the emission wavelength is determined primarily by the size of the quantum dots. This allows the absorption and emission wavelength to be further apart than for dye-doped dielectric nanospheres 150. This provides an extra degree of freedom in addition to the thickness of metal coating 145 in designing metal coated dielectric nanospheres 150. Because the emission and absorption processes are closely related, the absorption efficiency of the incident pump radiation may be increased resulting in an improvement in the emission efficiency.
Quantum dot, for example, ZnS-capped CdSe nanocrystals, doped dielectric nanospheres have been described by M. Han et al. in Nature Biotechnology, vol. 19, 631-635 (2001) and incorporated herein by reference. In particular, the dielectric material may be polystyrene or polymer. Doping by quantum dots is accomplished by swelling polystyrene nanospheres 150 in a solvent mixture containing 5 percent chloroform and 95 percent propanol or butanol and by then adding a controlled amount of ZnS-capped CdSe quantum dots. The doping process is typically complete in about 30 minutes at room temperature.
The first step in the metal coating process to make half metal coated doped dielectric nanospheres 150 is to disperse doped dielectric nanospheres 150 on a flat surface such as, for example, a glass slide. Polystyrene and latex nanospheres available from MOLECULAR PROBES CORPORATION are surfactant free and do not aggregate and are dispersed in a buffer fluid. Nanospheres of different sizes conjugated to biotin, avidin and streptavidin can be obtained from MOLECULAR PROBES CORPORATION. After spin coating and evaporation of the buffer fluid, the glass slide is coated with a monolayer of dielectric nanospheres 150. The glass slide is then introduced into a sputtering system. In order to improve metal adhesion which is especially important when using gold coatings, a layer of Ti or Cr is sputter deposited to a thickness in the range from about 2 nm to about 3 nm. This is followed by a sputter deposition of metal coating 145 to the desired thickness. Because the top half of dielectric nanospheres 150 block sputter deposition of the metal on the lower half of dielectric nanospheres 150, dielectric nanospheres 150 are typically half metal coated. Sputtering provides metal coating 145 with a high degree of metal uniformity. To achieve sufficient thickness for metal coating 145 on the sides it may be necessary to increase the metal thickness on the top. After the metal coating step, the glass slide is soaked in a suitable organic solvent, for example, propanol, to get half coated doped dielectric nanospheres 150 into solution for subsequent use. Ultra-sonification may be used if necessary to assist in removing half coated doped dielectric nanospheres 150 from the glass slide.
The attaching of metal specific molecules can be performed using molecules that have a terminal attaching group, for example thiol for gold, with a strong affinity to metal coating 145, 545 and a long linear alkyl chain that provides upright ordering on functionalized surface 410 or 555. Affinity to the solid surface is provided by a charge-transfer complex as in alkyl thiols on noble metals as described by Porter et al. in the Journal of the American Chemical Society, 109, 3559, (1987); Bain et al. in Angewandte Chemie, International Edition, 28, 506, (1989); and Nuzzo in the Journal of the American Chemical Society, 112, 558, (1990), all incorporated herein by reference.
For SiO2, the affinity to dielectric portion of half metal coated dielectric nanospheres 150 or nanoparticle such as nanoellipsoid 550 is provided by a covalent chemical reaction, for example, silanes as described by Sagiv in the Journal of the American Chemical Society, 102, 92, 1980; Wasserman et al. in Langmuir, 5, 1074, (1989); and Ulman in Angewandte Chemie Advanced Materials, 2, 573, (1990), all incorporated by reference herein.
While the invention has been described in conjunction with specific embodiments, it is evident to those skilled in the art that many alternatives, modifications, and variations will be apparent in light of the foregoing description. Accordingly, the invention is intended to embrace all other such alternatives, modifications, and variations that fall within the spirit and scope of the appended claims.
