Patent Grant
7867770
The fields of nanoscience and nanotechnology generally concern the synthesis, fabrication and use of nanoparticles and nanostructures at atomic, molecular and supramolecular levels. The nanosize of these particles and structures offers potential for research and applications across various scientific disciplines such as materials science, engineering, physics, chemistry, spectroscopy, computer science, microscopy and biology. For example, surfaces or substrates employing nanostructures can be used to enhance Raman scattering by many orders of magnitude, an effect often referred to as surface enhanced Raman scattering (SERS).
SERS is a common spectroscopic technique that can be used for detecting and identifying biological molecules. Typically, unique vibrational signatures or fingerprints can be observed for biological molecules via SERS. In aqueous media, the ability to rapidly produce such a vibrational signature for a biological molecule lacking visible chromophores demonstrates the potential of SERS as a valuable analytical and structural spectroscopic technique. SERS can be particularly useful for the detection and identification of biological molecules present in a sample at low concentrations. Recent SERS applications and developments have also extended toward the detection and identification of bacterial and viral pathogens. To date, conventional surfaces or substrates for SERS have been largely unsuccessful in reproducibly and reliably detecting and identifying such pathogens.
The present invention provides a nanostructured substrate for SERS. In one embodiment, a substrate of the invention comprises a surface featuring substantially monodisperse-sized metal nanoparticles disposed thereon. Preferably, the substrate surface provides for SERS and spectra therefrom generally referred to as SERS spectra. For example, a nanostructured substrate of the invention can be used to reproducibly and reliably detect or identify pathogens, molecules or combinations thereof. A substrate of the invention overcomes the shortcomings of conventional surfaces or substrates for SERS such as those described above.
A substrate of the invention can comprise monodisperse-sized metal nanoparticles substantially aggregated in clusters. Exemplary clusters of monodisperse-sized metal nanoparticles can comprise from about 2 to 25 nanoparticles. In one embodiment, nanoparticles for a substrate of the invention are substantially spheroidal comprising diameters in a range from about 40 to 120 nanometers (nm). Preferably, metal nanoparticles can comprise diameters in a range from about 80 to 120 nanometers nm. Metal nanoparticles for a substrate of the invention can also comprise silver, gold, copper or combinations thereof. A nanostructured substrate of the invention can comprise silicon, aluminum, titanium or combinations thereof.
Preferably, a substrate of the invention provides for SERS of an entity. Exemplary entities can include spores, pathogens, fluids, cells, amino acids, biological materials, molecules, nucleic acids, tissue samples, viruses, bacteria, inorganic materials, serums, vegetative samples, germinating samples, sputum, human blood, bronchoalveolar lavage fluid, cerebral spinal fluid or combinations thereof. In one embodiment, a nanostructured substrate can comprise an entity substantially disposed on a surface thereof. The substrate surface also comprises substantially monodisperse-sized metal nanoparticles. The entity of the substrate can be in contact with one or more substantially monodisperse-sized metal nanoparticles.
The invention also provides a method of detecting or identifying an entity via a nanostructured substrate comprising monodisperse-sized metal nanoparticles disposed on a surface thereof. In one embodiment, the method comprises providing a nanostructured substrate of the invention. The method also comprises disposing an entity substantially on the surface of the substrate. Preferably, the method comprises performing Raman microscopy of the entity on the substrate surface. Exemplary Raman microscopy yields at least one SERS spectrum of the entity. The method of the invention can also comprise detecting or identifying the entity based on a SERS spectrum.
For example, a method of the invention can detect or identify an entity by a comparison of the SERS spectrum to a reference vibrational signature. In one embodiment, a reference vibrational signature for an entity can be part of a vibrational signature library. Preferably, a vibrational signature library comprises SERS spectra for a plurality of entities. An exemplary vibrational signature library can be produced or developed using a nanostructured substrate of the invention for SERS. A vibrational signature library of the invention can comprise at least one reference vibrational signature.
In another embodiment, the invention provides a vibrational signature for an entity. Preferably, the vibrational signature is obtained by providing a nanostructured substrate comprising monodisperse-sized metal nanoparticles. An entity can be disposed on the surface of the substrate for performing Raman microscopy thereof. Exemplary Raman microscopy yields at least one SERS spectrum, which comprises a vibrational signature unique to the entity. For example, the vibrational signature can be a reference vibrational signature for detecting or identifying the entity. The vibrational signature can also be part of a vibrational signature library comprising SERS spectra for a plurality of entities such as pathogens, molecules or combinations thereof.
The invention also provides a method for detecting or identifying an entity via SERS spectra. In one embodiment, the method comprises providing a nanostructured substrate featuring monodisperse-sized metal nanoparticles disposed on a surface thereof. The method also comprises disposing an entity substantially on the surface of the substrate. Preferably, the method comprises performing Raman microscopy of the entity on the substrate surface to yield at least one SERS spectrum of the entity. Preferably, the entity disposed on the surface of the substrate is in contact with one or more substantially monodisperse-sized metal nanoparticles.
The method of the invention also comprises comparing a SERS spectrum of the entity to spectra of a vibrational signature library. As described above, an exemplary vibrational signature library can comprise SERS spectra for a plurality of entities. For example, the vibrational signature library can comprise SERS spectra of pathogens such as bacteria or viruses. Preferably, the method of the invention comprises detecting or identifying an entity based on a comparison of at least one SERS spectrum thereof to spectra of the vibrational signature library.
The invention also provides methods for syntheses of a nanostructured substrate comprising substantially monodisperse-sized metal nanoparticles. In one embodiment, a method for synthesis of a nanostructured substrate for SERS comprises hydrolyzing a solution comprising metal precursors. Moreover, the method comprises reducing the metal precursors in a matrix for subsequent growth of substantially monodisperse-sized metal nanoparticles on a surface of the substrate, which forms from the matrix. The method also comprises further reducing the metal precursors to grow the metal nanoparticles on the substrate surface.
Preferably, the metal precursors are rapidly reduced in the matrix during a first reduction step. The metal precursors are then slowly reduced during a second reduction step to grow substantially monodisperse-sized metal nanoparticles on the surface of the substrate. In one embodiment, the first reduction step can also be characterized by seed growth in the matrix. These seeds provide preferential growth sites for the subsequent growth of monodisperse-sized metal nanoparticles on a surface of the substrate. The second reduction step can also aid in the grow of the seeds within the matrix.
The invention also provides methods for uses of a nanostructured substrate comprising substantially monodisperse-sized metal nanoparticles. In one embodiment, a method for using a nanostructured substrate of the invention comprises obtaining SERS spectra therefrom. Preferably, the SERS spectra comprise a vibrational signature unique to at least one entity such as a pathogen, molecule or combination thereof. The vibrational signature of the spectra can also be a used to detect and identify the entity. For example, the entity can be detected or identified in any suitable media such as an aqueous media.
Other features and advantages of the invention may also be apparent from the following detailed description thereof, taken in conjunction with the accompanying drawings of which:
a) shows SERS spectra for B. subtilis spores produced by using a nanostructured substrate of the invention; and
b) shows SERS spectra of the supernatant from culture medium containing bovine viral diarrhea virus particles and a SERS spectrum for the corresponding culture medium supernatant of non-infected cells obtained via a nanostructured substrate of the invention.
Unless otherwise stated, the following definitions provide meaning and examples to terms used herein. Such definitions are also intended to encompass any meaning that may be contemplated by a person of ordinary skill within the art.
The terms “monodisperse” or “monodisperse-sized” and derivations thereof such as substantially monodisperse-sized generally refer to substantially uniformly or homogeneously proportioned or sized particles such as metal nanoparticles. For example, metal nanoparticles for a nanostructured substrate of the invention can be physically distinguishable from particles within conventional surfaces or substrates for SERS as these nanoparticles are substantially uniformly or homogeneously proportioned or sized.
The present invention provides a nanostructured substrate for SERS. A substrate of the invention comprises a surface featuring substantially monodisperse-sized metal nanoparticles disposed thereon. In one embodiment, a nanostructured substrate of the invention comprises a surface featuring metal nanoparticles substantially aggregated in clusters. Exemplary metal nanoparticles disposed on the substrate surface can be silver, gold, copper or combinations thereof. Preferably, metal nanoparticles disposed on a substrate surface can be substantially spheroidal. A substrate surface can also comprise silicon dioxide, aluminum oxide, titanium dioxide or combinations thereof.
The invention also provides methods for syntheses or uses of a nanostructured substrate comprising monodisperse-sized metal nanoparticles disposed on a surface thereof. In one embodiment, a method of the invention can be performed to obtain a nanostructured substrate comprising monodisperse-sized metal nanoparticles. For example, the method can comprise growing substantially monodisperse-sized metal nanoparticles on the substrate surface. Preferably, a method of the invention is also performed to detect or identify an entity. The method can comprise performing Raman microscopy of the entity to produce a SERS spectrum thereof.
The surface topology or morphology of exemplary substrates such as shown in
SERS and spectra therefrom via a substrate of the invention can be comparable for different types of nanoparticles, although vibrational signatures of an entity are generally metal dependent. Vibrational signature metal dependence for a substrate can be related to different surface topologies or morphologies. In addition, vibrational signature metal dependence can also be related to the chemical properties of the nanoparticles disposed on the substrate surface. Preferably, a substrate of the invention can provide for SERS spectra of bacterial or viral pathogens. A nanostructured substrate can also yield SERS spectra of chemical or biological molecules. For example, a substrate of the invention provides for a Raman cross-sectional enhancement of about 5×107 for glycine at an excitation of 785 nm.
In one embodiment, a substrate of the invention comprises an entity substantially disposed on a surface thereof. The substrate surface also comprises monodisperse-sized metal nanoparticles.
Exemplary entities for a substrate of the invention can include spores, pathogens, fluids, cells, amino acids, biological materials, molecules, nucleic acids, tissue samples, viruses, bacteria, inorganic materials, serums, vegetative samples, germinating samples, sputum, human blood, bronchoalveolar lavage fluid, cerebral spinal fluid or combinations thereof. In one embodiment, a nanostructured substrate can provide for SERS and spectra therefrom, which are unique to an entity. Preferably, the entity can be in contact with one or more monodisperse-sized metal nanoparticles substantially disposed on the substrate surface.
The examples herein are provided to illustrate advantages of the invention that have not been previously described. The examples are also intended to assist a person of ordinary skill within the art in syntheses and uses of a nanostructured substrate of the invention. The examples can incorporate or otherwise include any variations or inventive embodiments or aspects as described above. The embodiments described above can also incorporate or otherwise include any variations or inventive embodiments or aspects of the examples herein. The examples are also not intended in any way to limit or otherwise narrow the disclosure or scope thereof as generally provided herein.
Exemplary syntheses of nanostructured substrates of the invention were performed via in situ growth methods. For example, a gold ion doped sol-gel was formed by hydrolysis of tetramethoxysilane (Si(OCH3)4) in an acidic (about 0.005 milliliters (ml) of about 1 percent (%), volume per volume, concentration of hydrochloric acid (HCl)) methanol solution (about 10 ml of high performance liquid chromatography (HPLC) grade methanol, about 5 ml of water and about 3 ml of Si(OCH3)4 99.99% from Sigma-Aldrich, St. Louis, Mo. 63103) of metal precursors such as chlorauric acid (HAuCl4) (about 50 microliters (μl) of about 1 molar (M) HAuCl4 from Sigma-Aldrich) based metal precursors.
After about 3 hours of agitation to complete hydrolysis, sol-gel aliquots (about 25 μl) within microcentrifuge tubes, such as polypropylene microcentrifuge tubes, were dried in a fume hood for about 12 to 48 hours at ambient temperature and airflow (relative humidity about 40%). The resulting matrixes of gel pellets or chips comprising metal precursors were then exposed to water saturated air for about 1 hour. These gel pellet or chip matrixes were vigorously agitated (about 30 seconds) with about 0.66 millimolar (mM) of a reducing agent such as an aqueous sodium borohydride (99.99% from Sigma-Aldrich) solution in a first reduction step. The first reduction step rapidly reduced the metal precursors in the matrixes providing gold seeds for substantially monodisperse-sized metal nanoparticle surface growth during a second reduction step.
The solution was drained and about 50 ml of water were added to the gel pellet or chip matrixes. Gentle agitation was then induced for about 30 minutes to form silicon dioxide substrates from the matrixes for monodisperse-sized metal nanoparticle growth thereon. For the second reduction step, these substrates remained in a low concentration of a reducing agent for about 24 hours. The second reduction step slowly reduced the metal precursors to grow monodisperse-sized gold nanoparticles substantially on an exposed outer surface of the substrate, yielding an exemplary nanostructured substrate of the invention such as shown in
To aid in consistent syntheses of nanostructured substrates of the invention, ambient airflow can also be filtered via a hydrocarbon absorbing filter and 300 micron (μ) particulate filters. The exemplary nanostructured substrates of the invention provide for SERS. The invention also contemplates performing exemplary syntheses or variations thereof to yield substantially monodisperse-sized metal nanoparticles other than gold. For example, given syntheses can be varied to produce monodisperse-sized silver nanoparticles substantially disposed on a substrate of the invention by substituting silver nitrate (AgNO3) for HAuCl4, limiting the first reduction step to about 5 seconds and employing about a five-fold lower reducing agent concentration.
Exemplary syntheses of a nanostructured substrate can also be varied by manipulating metal precursor or reducing agent concentrations. Moreover, adjusting relative humidity during syntheses can yield low or high densities of substantially monodisperse-sized metal nanoparticles. For example, a low density of monodisperse-sized metal nanoparticles can partially cover or coat a surface for a nanostructured substrate of the invention. By comparison, a high density of monodisperse-sized metal nanoparticles can substantially or entirely cover or coat a substrate surface. Such variations to exemplary syntheses for a substrate of the invention can also affect Raman cross-sectional enhancement including SERS intensities.
Nanostructured substrates produced by exemplary syntheses can also feature monodisperse-sized metal nanoparticles substantially aggregated in clusters. For example, monodisperse-sized gold nanoparticles were substantially disposed on substrates comprising silicon dioxide, partially covering or coating outer surfaces thereof. In contrast to nanostructured substrates of the invention, conventional surfaces or substrates for SERS consist of metal particles or colloids embedded or dispersed therein.1 Metal particles embedded within conventional surfaces or substrates also tend to be nonuniformly or inhomogeneously proportioned and sized.2
Exemplary uses for nanostructured substrates include providing for SERS of entities such as bacterial or viral pathogens. A nanostructured substrate can also provide for SERS of chemical or biological molecules. To demonstrate SERS of bacterial pathogens, E. coli, S. typhimurium, Bacillus cereus (B. cereus), B. anthracis Sterne and B. thuringiensis samples were obtained from Carolina Biological Supply, Burlington, N.C. 27215. Bacillus subtilis (B. subtilis) YS11 was also obtained from the Bacillus Genetic Stock Center (BGSC), Columbus, Ohio 43210.
In addition, B. subtilis 3610 (SSB2) and its congenic insertion deletion construct hag::erm (SSB71) were provided by the Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Mass. 02115. A B. anthracis Sterne cotE::cat mutant was also provided by the Department of Microbiology and Immunology, Loyola University Medical Center, Chicago, Ill. 60153. The in vitro bacterial pathogen samples were grown in about 5 ml of Luria-Bertani (LB) broth (about 5 hours) to an OD600 equal to about 1. The samples were then washed five times with water and resuspended in about 0.25 ml of water.
A platinum loop was used to place about 1 μl of a bacterial pathogen sample suspension on an nanostructured substrate of the invention. SERS spectra were generally acquired within minutes after placing the samples on the substrate surface comprising substantially monodisperse-sized metal nanoparticles. Bulk Raman spectra of bacterial pathogen samples, excited with about 300 milliwatts (mW) at about 785 nm, were correspondingly placed on a standard potassium bromide (KBr) material. A Renishaw Raman microscope (model RM2000) capable of about 2λ spatial resolution was used for measurements at about 785 nm (diode laser excited).
Typically, SERS spectra of bacterial pathogen samples on a nanostructured substrate of the invention were obtained with an incident laser power at about 1 to 3 mW and spectral acquisition time of about 10 seconds. An exemplary spectral resolution was set to about 3 cm−1 for a cooled charged coupled device (CCD) (400×578 array size) detection system (0.25 meter (m) spectrometer fitted with a 1200 groove per millimeter (mm) grating). A 520 cm−1 vibrational band of a silicon wafer provided frequency calibration. Several microscope objectives including about 50 and 100 times (×) objectives were used for excitation and collection.
Correspondingly, Raman cross-sectional enhancement per B. anthracis Sterne bacterium was about 5×104 due to the gold nanoparticles of the substrate. The greater amplification factor for B. anthracis Sterne as compared to E. coli may be attributable to different Gram-positive and Gram-negative cell surface structures of these two types of bacterial pathogens. About 300 mW of incident 785 nm excitation power and 100 second signal accumulation times were used to obtain the bulk Raman spectra of
The bulk Raman spectrum of B. anthracis Sterne excited at 785 nm was dominated by broad fluorescence as shown by spectrum (d) in
The absolute scattering intensities of these spectra varied by less than about 15% at about 735 cm−1 (signal maximum). These spectra were typical of samples from the same B. anthracis Sterne culture or cultures grown on different days in a common broth type. The spectra in
In addition to enhanced species vibrational specificity, the number of transitions in the SERS spectra of E. coli and S. typhimurium via a substrate of the invention were significantly fewer than in corresponding bulk Raman spectra.
Additionally, the spectra of
For example, a vibrational signature library of the invention can be maintained on a system comprising a processor such as computer. Such a computer can also comprise algorithms or instructions that are stored in memory and executed by the processor for the detection or identification of an entity by using a substrate of the invention for SERS. The invention also contemplates that exemplary algorithms or instructions can be performed by a processor executing scripts, compiled programs or any other suitable components such as downloadable applets or plug-ins. Furthermore, a vibrational signature library can be stored on firmware, hardware, software or combinations thereof such as combinatorial logic, integrated circuits or gate arrays.
Using a nanostructured substrate of the invention for SERS, bacterial pathogen strains and mutants can also be distinguished based on unique vibrational signatures. For example, SERS spectra for a B. subtilis congenic mutant lacking flagella hag::erm and B. subtilis strains, YS11 and 3610, exhibited distinct vibrational signatures using a nanostructured substrate of the invention. These spectra demonstrated that a nanostructured substrate can provide a reproducible and reliable basis for detection or identification of closely related species of bacterial pathogens.
Additional evidence for the ability of a nanostructured substrate of the invention to provide species specific vibrational signatures for detection and identification of bacterial pathogens is demonstrated by
As an example of single cell capability for a substrate of the invention, SERS spectrum of a B. anthracis Sterne (cotE::cat) mutant two-cell chain was detected and identified. In particular,
The SERS spectrum of multiple B. anthracis cells (about 30 cells) was also obtained with less tightly focused excitation (50× objective and about 10 seconds for data accumulation). In addition to enabling bacterial pathogen mixture identification, single cell detection capabilities for a substrate of the invention can minimize the effects of spectral contamination in the SERS of biological fluids such as a fluid comprising an entity of interest. Moreover, spectral contributions from non-bacterial components of in vivo derived samples can be greatly reduced as a result of the ability to observe vibrational signatures from a bacterium filled sampling volume.
a) shows SERS spectra for B. subtilis spores provided via a nanostructured substrate of the invention. An aqueous suspension of B. subtilis spores were placed on the substrate. The substrate comprised a surface featuring substantially monodisperse-sized silver nanoparticles. The SERS spectra of these spores were readily observed in about 20 seconds with about 2 mW of 785 nm excitation.
b) shows SERS spectra of the supernatant from culture medium containing bovine viral diarrhea virus particles (upper trace) and a SERS spectrum for the corresponding culture medium supernatant of non-infected cells (lower trace) produced by using a nanostructured substrate of the invention. The nanostructured substrate comprising a surface featuring substantially monodisperse-sized gold nanoparticles. The differences between the upper and lower trace in
While the invention has been described herein in conjunction with a preferred embodiment, a person of ordinary skill within the art, in view of the foregoing, can effect changes, substitutions of equivalents and other types of alterations to the nanostructured substrates for SERS set forth herein. Each embodiment described above can also have incorporated or otherwise included therewith such variations as disclosed in regard to any or all other embodiments. Thus, it is intended that protection granted by Letter Patent hereon be limited in breadth and scope only by definitions contained in the appended claims and any equivalents thereof.
This application claims the priority of U.S. Provisional Application No. 60/632,930 filed Dec. 3, 2004 and entitled, SEEDED DEPOSITION OF METAL NANO-CLUSTERS USING NANO-PARTICLES ON A SOLID MATRIX, and U.S. Provisional Application No. 60/633,735 filed Dec. 6, 2004 and entitled, SEEDED DEPOSITION OF METAL NANO-CLUSTERS USING NANO-PARTICLES ON A SOLID MATRIX, which are hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2005/043793 | 12/5/2005 | WO | 00 | 5/31/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2006/060734 | 6/8/2006 | WO | A |
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| 6180415 | Schultz et al. | Jan 2001 | B1 |
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| 6514767 | Natan | Feb 2003 | B1 |
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| Number | Date | Country |
|---|---|---|
| WO 03010511 | Feb 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20080096005 A1 | Apr 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60632930 | Dec 2004 | US | |
| 60633735 | Dec 2004 | US |
