JIANG, Peng (8559 Sw 11th Road, Gainesville, FL, 32607, US)
BATICH, Christopher, D. (3733 Nw 40th Street, Gainesville, FL, 32607, US)
SCHULTZ, Gregory (832 Nw 45th Terrace, Gainesville, FL, 32605, US)
LIN, Tzung-hua (3700 Windmeadows Blvd. Apt. E60, Gainesville, FL 5, 326085, US)
CHUNG, Pei-yu (3700 Windmeadows Blvd. Apt. S194, Gainesville, FL, 32608, US)
JIANG, Peng (8559 Sw 11th Road, Gainesville, FL, 32607, US)
BATICH, Christopher, D. (3733 Nw 40th Street, Gainesville, FL, 32607, US)
SCHULTZ, Gregory (832 Nw 45th Terrace, Gainesville, FL, 32605, US)
LIN, Tzung-hua (3700 Windmeadows Blvd. Apt. E60, Gainesville, FL 5, 326085, US)
| CLAIMS The invention claimed is: 1 . A surface plasmon sensor comprising an array of shaped protuberances having a metallic surface. 2. The sensor of claim 1 , wherein the shaped protuberances have a dimpled top surface. 3. The sensor of claim 1 , wherein the array comprises at least a first layer and a second layer of shaped protuberances, and wherein the shaped protuberances in the first and second layers comprise dimpled top surfaces. 4. The sensor of claim 1 , wherein the shaped protuberances have a tapered shape. 5, The sensor of claim 4, wherein the shaped protuberances have a substantially pyramidal shape. 6. The sensor of claim 1 , wherein the shaped protuberances comprise a plurality of spaced apart protuberances. 7. The sensor of claim 1 , wherein the shaped protuberances comprise a silica-polymer composite. 8. The sensor of claim 1 , wherein the shaped protuberances are disposed on a substrate, and wherein the substrate comprises a member from the group consisting of silicon, quartz, glass, a polymer, and combinations thereof. 9. The sensor of claim 8, wherein the substrate is a planar substrate. 10. The sensor of claim 8, wherein the substrate is an optical fiber. 1 1 . The sensor of claim 1 , wherein the metallic surface comprises a member selected from the group consisting of gold, silver, aluminum, copper, titanium, chromium, and combinations thereof. 12. A surface plasmon sensor comprising an array of shaped protuberances having a dimpled and a metallic top surface. 13. The sensor of claim 12, wherein the shaped protuberances are formed from a polymer. 14. The surface plasmon sensor of claim 12, wherein the metallic top surface comprises a member selected from the group consisting of gold, silver, aluminum, copper, titanium, chromium, and combinations thereof. 15. The surface plasmon sensor of claim 12, wherein the shaped protuberances are nanosized protuberances. 16. A method for making a surface plasmon sensor, the method comprising: preparing a colloidal suspension comprising silica and a polymer; spin coating the colloidal suspension to form crystal colloids in the polymer; and etching to selectively remove polymer to provide an array of dimpled protuberances comprising the crystal colloids; and depositing a metallic layer over the dimpled protuberances to form the sensor. 17. The method of claim 16, wherein the crystal colloids comprise silica. 18. The method of claim 16, wherein the etching is done by oxygen plasma etching, and wherein the polymer comprises a non-volatile acrylate monomer. 19. The method of claim 16, wherein the etching is done from 15 to 105 seconds. 20. The method of claim 19, wherein the etching is done from 90 to 105 seconds. 21 . The method of claim 16, wherein the dimpled protuberances comprise nanoparticles. 22. A method for making a surface plasmon sensor comprising: depositing a polymer material in a first substrate having a plurality of shaped recesses etched therein; removing the polymer material from the first substrate using a second substrate to provide a plurality of shaped protuberances on a surface of the second substrate; and depositing a layer of a metallic material over the plurality of shaped protuberances to form said sensor. 23. The method of claim 22, where the polymer material is polyurethane. 24. The method of claim 22, wherein the shaped protuberances are in the form of spaced apart nanopyramids. 25. The method of claim 22, wherein the shaped protuberances have a dimpled top surface. 26. The method of claim 22, wherein the first substrate is formed from silicon, and the second substrate is formed from one of glass or quartz. 27. The method of claim 22, wherein the first substrate is generated by a self- assembly method comprising: depositing silica particles and polymer on the surface of the first substrate; etching polymer and depositing a mask layer which produce a plurality of periodic nanohole arrays; and aniotropic wet etching through the plurality of periodic nanohole arrays to provide the tapered recesses. 28. The method of claim 22, wherein the mask layer is a metallic material deposited by electron-beam evaporation. 29. The method of claim 28, wherein the mask layer is of chromium. 30. The method of claim 23, wherein the metallic material comprises a member selected from the group consisting of gold, silver, aluminum, copper, titanium, chromium, and combinations thereof. 31 . The method of claim 30, wherein a thin layer of metallic material is continuous and has a thickness selected from the range of 10 nm to 100 nm. 32. A method for determining a presence of an analyte in a sample in a surface plasmon resonance system, said method comprising: introducing an energy source to excite surface plasmon resonance; flowing an analyte over a flow cell comprising a sensor having a plurality of shaped protuberances thereon; and combining signals from one or more detectors to detect a change in proximity of the sensor chip. 33. The method of claim 32, wherein the shaped protuberances comprise spaced apart nanopyramids. 34. The method of claim 32, wherein the shaped protuberances have a dimpled top surface. 35. The method of claim 32, wherein the shaped protuberances are functionalized with one or more agents that have an affinity for a target analyte. 36. The method of claim 35, wherein the agents comprise a member from the group consisting of proteins, oligonucleotides, DNA molecules, RNA molecules, lipids, carbohydrates, glycoproteins, lipoproteins, peptides, polysaccharides, polypeptides, and chemical entities. 37. The method of claim 32, wherein the one or more detectors receive electromagnetic radiation transmitted, scattered, absorbed or reflected by the sensor and sense spectrum and image changes. 38. The method of claim 37, wherein the spectrum and image changes are reflected in changes in frequency, intensity, phase, or a characteristic of opto-electrical signals. 39. The method of claim 37, wherein said one or more detectors comprise a surface enhanced Raman spectroscopy (SERS) detector, a spectrometer, or an image detector. |
CROSS REFERENCE TO RELATED APPLICATIONS
This application is related to U.S. Provisional Application No. 61 /421 ,379 filed December 9, 2010, to which priority is claimed under 35 USC 1 19. This application is incorporated herein in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED DEVELOPMENT
Development for this invention was supported in part by Contract
No. U54NS058185, awarded by the National Institute of Health and administered through the Lovelave Respiratory Research Institute. Development for this invention was also supported in part by Contract No. CBET-0744879, awarded by the National Science Foundation and by Contract No. DTRAOI-03-D-0010, awarded by the Defense Threat Reduction Agency. Accordingly, the United States Government has certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates to sensors, and more particularly to surface plasmon sensors for the detection of target analytes of interest.
BACKGROUND OF THE INVENTION
Surface-enhanced Raman scattering (SERS) and surface plasmon resonance (SPR) detection are two key surface plasmon (SP) techniques that will ultimately enable single-molecule-level chemical and biological sensors. SERS is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. SPR refers to the excitation of surface plasmons by light. SERS and SPR sensors may be of great importance in the detection and identification of chemical and biological warfare agents, improvised explosive devices, and other asymmetrical warfare weapons. Colloidal nanoparticles (e.g., Au and Ag nanoparticles) are currently used in SERS and SPR detection. However, the stochastic aggregation of colloidal nanoparticles significantly affects the sensing reproducibility. Further, various periodic plasmonic nanostructures with well-defined SP properties have been broadly exploited. Unfortunately, the development and implementation of these nanostructured SP substrates for chemical and biological sensing applications have been greatly impeded by expensive and painstaking top-down nanofabrication (e.g., electron-beam
lithography and focused ion-beam), which limit the available sample size to less than 1 mm 2 .
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A is a tilted-view (35°) and includes SEM images of a templated gold nanopyramid array.
FIG. 1 B is a magnified image of the nanopyramid array of FIG. 1 A where 320 nm silica spheres were used as template.
FIG. 2 shows the normalized reflection of white light (incident angle of 45°) from a gold nanopyramid array immersed in glycerol solutions of different refractive indices.
FIG. 3A is a FDTD-simulated optical reflection of plane wave from a gold nanopyramid array with liquid of different refractive indices.
FIG. 3B is a comparison between experimental (solid) and FDTD-simulated optical reflection from nanopyramids with 10 nm (dashed) and 50 nm (dotted) tips.
FIG. 4 is a comparison of detection sensitivity between the experimental result and FDTD simulation.
FIG. 5 is a normalized reflection spectra obtained from a gold nanopyramid array with adsorbed anti-alcohol dehydrogenase (black), followed by the addition of specific alcohol dehydrogenase (red) and nonspecific protein, casein (blue).
FIGS. 6A-6D show SEM images of the templated polymer nanodimple arrays. These samples were prepared by oxygen plasma etching of a spin-coated colloidal crystal-polymer nanocomposite for different durations. (A) 15 sec; (B) 30 sec; (C) 90 sec; and (D) 105 sec.
FIGS. 7A-7D show SEM images of the resulting SERS and SPR substrates. These samples were also prepared by oxygen plasma etching of a spin-coated colloidal crystal-polymer nanocomposite for different durations. (A) 15 sec; (B) 30 sec; (C) 90 sec; and (D) 105 sec.
FIG. 8 shows SER spectra recorded for benzenethiol molecules adsorbed on templated Au nanodimples with different plasma etching durations. FIG. 9 shows reflectance spectra of templated Au nanodimples prepared by different oxygen plasma etching durations. The dashed line shows the position of the laser excitation wavelength.
FIG. 10 shows the optical reflection of white light (incident angle of 45°) from an Au nanodimple array (30 s etch) immersed in glycerol solutions of different refractive indices.
FIG. 1 1 shows the optical reflection of white light (incident angle of 45°) from an Au nanodimple array (60 s etch) immersed in glycerol solutions of different refractive indices.
FIG. 12 shows the optical reflection of white light (incident angle of 45°) from an
Au nanodimple array (120 s etch) immersed in glycerol solutions of different refractive indices.
FIG. 13 is a schematic of an SPR system in accordance with an aspect of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have advantageously developed chemical and biological sensors, systems and methods, which utilize periodic arrays having a plurality of shaped protuberances disposed on a substrate. As will be explained below, aspects of the present invention enable the label-free detection of chemical and biological molecules without regard to the size of the molecule. In addition, aspects of the present invention enable an increased enhancement factor and/or increased sensitivity for the sensors, systems and methods described herein. Further, the shaped protuberances described herein enable the excitement of both localized and propagating surface plasmons, which provide a highly sensitive and highly reproducible analytical tool for the detection of chemical entities and biomolecules in a sample. In one embodiment, for example, specific antigen-antibody binding can be detected by using a sensor having an array of shaped protuberances as described herein in a real-time and label-free manner.
In accordance with an aspect of the present invention, the shaped protuberances have a definite repeating structure. As shown in FIGS. 1 A-1 B, there is shown a first embodiment of a sensor 10 having an array of shaped protuberances 12 have a tapered profile 14 disposed across a top surface 16 of a substrate 1 8. By "tapered," it is meant a body having an upper portion having a cross-section with a smaller diameter than a diameter than a lower portion of the body. Thus, in one embodiment, the shaped protuberances 12 are substantially cone-shaped. In another embodiment, the shaped protuberances 12 are substantially pyramidal in shape. By "pyramidal," it is meant a polyhedron having a polygonal base and triangular sides with a common vertex. The sides of the pyramidal protuberances may have any desired angle of inclination. In one embodiment, the pyramidal protuberances have an angle of inclination of 45 or less. The shaped protuberances 12 each further include a metallic coating 20 over at least a portion of the shaped protuberances to form a metallic surface. In one embodiment, the shaped protuberances 12 are completely coated with the metallic coating 20. The metallic coating 20 may comprise gold, silver, aluminum, copper, titanium, chromium, and combinations thereof, for example. In addition, the metallic coating 20 may be applied using any suitable deposition technique known in the art, such as by known sputtering and electron-beam deposition techniques.
The shaped protuberances 12 may comprise a silica material underlying the metallic coating 20. In another embodiment, the shaped protuberances 1 2 may instead or further comprise a polymeric material, such as a polyurethane or an ethoxylated trimethylolpropane triacrylate monomer.ln a particular embodiment, the shaped protuberances 12 comprise a polymer-silica composite.
Referring again to FIG. 1 A, Fig. 1 A shows tilted scanning electron microscope (SEM) images of the shaped protuberances 12 in the form of an array of gold nanopyramids 22 templated from 320 nm silica spheres. The long-range hexagonal ordering of the nanopyramid array 22 can be clearly seen from Fig. 1 A. A magnified SEM image in FIG. 1 B reveals the sharp tips and the smoothness of the templated nanopyramids 22. Without wishing to be bound by theory, it is believed that the sharp tips of nanopyramid array 22 are particularly suitable for exciting strong surface plasmon resonance due to the efficient localization of an electromagnetic field. It is further believed that the nanopyramid arrays described herein can enable multiple spectroscopic techniques by allowing for the excitement of both localized and propagating surface plasmons. The multiple spectroscopic techniques may include Surface Enhanced Raman Scattering (SERS) Spectroscopy and spectrometry with detection ranges from ultraviolet (UV), visible (VIS) to near infrared (NIR). Typically, the spectrum modulation corresponds to the change and interaction at the surface of the sensor.
The substrate 18 may be formed from one or more of silicon, quartz, glass, a polymeric material, or any other suitable material. In one embodiment, the substrate 18 is formed from glass, which aids in obtaining transmission spectra from the sensor chip. In another embodiment, the substrate comprises a polymer. The substrate 18 may be planar or non-planar. Non-planar substrates include, but are not limited to, an optical fiber. The shaped protuberances 12 may be adjoined, affixed, or otherwise maintained on the substrate 18 by any suitable method known in the art. In one embodiment, the shaped protuberances 12 are maintained on the substrate 18 by a suitable binder adhesive as is known in the art. In another embodiment, the substrate 18 and the shaped protuberances 12 are integrally formed such that there is no distinction between the substrate 18 and the shaped protuberances 1 2.
In accordance with another aspect of the present invention, the shaped protuberances 12 may instead have a dimpled profile. As shown in FIGS. 6A-6D, for example, there is shown a sensor 100 comprising an array of shaped protuberances 102 thereon, each of the shaped protuberances 1 02 having a dimpled top surface 104 (hereinafter "dimpled protuberances 102"). The inventors have surprisingly found that the dimpled protuberances 102 provide SERS sensors with a very high enhancement factor (> 10 7 ) and/or provides highly sensitive SPR sensors. The localized of electromagnetic field contributes the electromagnetic 'hot spots' surrounding the dimpled surface. While not wishing not to be bound by theory, it is believed that the dimpled protuberances provide circulating waveguide modes resulting from the ring resonator. The dimpled protuberances may be formed from any suitable material as set forth above with the tapered protuberances, such as a polymer and/or silica. In one embodiment, the dimpled protuberances 102 comprise a colloidal crystal-polymer nanocomposite. The colloidal crystals may be silica or silica-based, for example.
In addition, the dimpled protuberances 102 may comprise a single layer 106 of dimpled protuberances 102 as shown in FIGS. 6A-6D. In another embodiment, as shown in FIGS. 6C-6D, the dimpled protuberances102 comprises the first layer 1 06 of dimpled protuberances 102 and a second layer 1 08 of dimpled protuberances 1 02. The addition of the second layer 1 08 of the dimpled protuberances 102 may be a result of a longer etching time as described below. It is contemplated that the dimpled
protuberances 102 may be of any suitable depth capable of increasing an enhancement factor or sensitivity of the associated sensor relative to a sensor without the dimpled protuberances 102.
In a particular embodiment, the dimpled protuberances 102 are formed by fabricating the dimpled protuberances 102 using a spin-coating technique. The methodology is based on shear-aligning concentrated colloidal suspensions using standard spin-coating equipment to form a colloidal crystal-polymer nanocomposite. During the spin-coating process, neighboring spheres tend to move to the vacancies to keep the continuity of the film, resulting in a convective radial particle flow, which leads to a pressure gradient exerted normal to the free film surface. In one embodiment, the colloidal suspensions comprise solid particles dispersed within a polymer. In a particular embodiment, the solid particles comprise silica and the polymer comprises a non-volatile acrylate monomer. Advantageously, the formation of colloidal crystals from the colloidal suspensions occurs within seconds as indicated by the appearance of a striking diffraction star with six arms. This diffraction star pattern is characteristic of long-range hexagonal ordering. In one embodiment, the spin-coating technique enables mass-fabrication of wafer-scale (e.g., up to 12 inch) colloidal crystals, which is a length scale nearly three-orders of magnitude larger than currently available through other methods. In addition, the colloidal crystals may be formed within minutes, as compared to days or even weeks needed to produce a centimeter-sized crystal using known techniques.
Once the colloidal crystals are formed, oxygen plasma etching is used to selectively remove the top polymer layer on the colloidal crystals to release the embedded silica particles. Depending on the etching duration, the penetration depth of silica particles within the polymer can be precisely controlled. The exposed solid, e.g., silica, particles can then be dissolved in an acidic solution, such as a 2% hydrofluoric acid aqueous solution. The hydrofluoric acid is effective to selectively remove embedded silica spheres and generate large-area, flexible and free-standing
macroporous polymers. Referring again to FIGS. 6A-6D, FIG. 1 shows scanning electron microscope (SEM) images of the resulting dimpled protuberances 102 with different oxygen plasma etching durations. In one embodiment, the dimpled protuberances 1 02 are nanosized and thus comprise nanodimples 1 1 0. When the plasma etching time is short, only the top layer silica particles are exposed, resulting in the formation of a single layer of the nanodimples 1 1 0. When the plasma etching time is long, both the first (e.g., top) layer 106 and the second layer 1 08 of silica particles are exposed, thereby resulting in the formation of binary nanodimples having the first layer 106 and the second layer 108 of dimpled protuberances 1 10. A thin layer of the metallic coating 20 can then be deposited on the nanodimples 1 10 to form the final SERS and SPR substrates as shown in FIGS. 7A-7D. The metallic coating 20 may comprise gold, silver, aluminum, copper, titanium, and chromium, or combinations thereof. The coating 20 for any of the embodiments described herein may be of any suitable thickness, such as from 10-150 nm. In one embodiment, the coating is about (+ or - 1 0%) 100 nm in thickness.
The shaped protuberances (12, 102) may be spaced from one another by any suitable spacing. Advantageously, aspects of the present invention control the spacing between adjacent protuberances (e.g., nanopyramids) to provide a sensor having high sensitivity, as well as high reproducibility. Critically, the shaped protuberances 12, 1 02 have a reproducible and uniform structure and do not agglomerate as with prior art nanosensors. In one embodiment, the shaped protuberances 12,102 are spaced apart from one another by a length, which is less than a length of a base of an adjacent protuberance. Further, the shaped protuberances 12, 102 may be any suitable size for enabling the generation of surface plasmons. In one embodiment, the shaped protuberances 12, 102 have at least one dimension that is nanosized, e.g., from 1 -1 000 nm in size. For example, one or more of the shaped protuberances 12, 102 may have a dimension, e.g., a height from a surface of the substrate, which is 1 000 nm or less. In a particular embodiment, the shaped protuberances 12, 1 02 have a height of from 10-200 nm, although the present invention is understood to be not so limited.
In accordance with another aspect of the present invention, the array of shaped protuberances 12, 102 as described herein may be functionalized with one or more agents that have an affinity for a target analyte. Exemplary agents include one or more biomolecules or chemical entities. By "biomolecule," it is meant an organic molecule that may be found in a living organism or synthetically produced. Exemplary
biomolecules may include oligonucleotides, polynucleotides, oligopeptides, peptides, polysacharides and polypeptides having a complementary counterpart. Exemplary analytes of interest corresponding to the agents may also include one or more biomolecules or chemical entities.
The agents may be maintained on the metallic protuberance surface by any suitable method such as by adsorption, hydrogen boding, ionic bonding, covalent bonding, or the like. In one embodiment, a thiol-group may be introduced or conjugated on the agents such that thiol-group can covalently bind to the gold surface. In another embodiment, a cross-linking agent may be used, such as an agent having a thiol-group and which is amine- or carboxy-group terminated, such that the crosslinker could link the agent to the gold surface. In yet another embodiment, dextran may be used as a porous gel layer on the gold surface to absorb the agent on the gold surface.
The applications of the sensors described herein for recognition and
quantification of one or more target analytes is without limitation. In one embodiment, the agent comprises a biorecognition coating that interacts with the target molecules in a sample. While the molecules are absorbed, deabsorbed, cleaved, or modified on the surface, the change of the dielectric layer on the surface will cause modulation of sensor output. The modulation of light used as a sensor output could be angular, wavelength, intensity, phase, or polarization change. This technology can be used for analyzing kinetic and thermodynamic interaction of biomolecules including proteins, peptides, oligonucleotides, for example. The sensors described herein can also be used for any other chemical or physical sensing while the transducing medium is altered by analytes or reacts with target analytes. Due to the strong concentration of the electromagnetic field and the nanometer penetration depth in the dielectric, any variation in the dielectric adjacent to the interface will produce highly sensitive signals. Advantageously, the sensor chips as described herein enable highly reproducible and sensitive detection.
In accordance with another aspect of the present invention, the sensors, e.g., sensors 10, 1 00, can be incorporated into a surface plasmon resonance (SPR) system 200 as is known in the art. The SPR system 200 typically includes an energy source 202, a flow cell 204 comprising the sensor 10, 100 and a sample flow path 206 there through, a detector 208, as well as a data analysis system 210. The energy source 202 may be a white light source, such as a tungsten, deuterium, or xenon lamp.
Alternatively, the energy source 202 may include a light-emitting diode, a laser diode source, or any other suitable energy source. A flow path 206 may be provided to transmit the energy from the energy source 202 to the flow cell 204 as is necessarily. The flow cell 204 containing the sensor 10, 100 as described herein will typically include the flow path 206, which has a passageway 212 for flow of the analyte there through in a suitable medium. In one embodiment, the flow path 206 consists of hollow core tubing and embedded channels on a microfluidic device. The sensor 10, 100 may be disposed within the flow path 206 with one side of the sensor 10, 100 exposed to a sample fluid inlet 214 while an opposed side is disposed adjacent to a sample fluid outlet 216. The side that receives the energy from the energy source could be parallel or perpendicular to the sample flow direction.
It is contemplated that the sample will be introduced upstream of the sensor 10, 100 using any suitable sample injection device or technique known in the art. A pump (not shown) may be provided to drive the sample through the flow cell. The detector 208 may determine the extent of change of an opto-electrical signal which may include an image and/or spectroscopic response. In one embodiment, the detector 208 may be a photomuliplier, a photodiode, a charged-couple detector (CCD), a complimentary metal oxide semiconductor (CMOS) or spectroscopy. Any suitable opto-electronic devices and systems may be utilized for obtaining spectrometric data or images from the flow cell prior to, during, and/or after introduction of a sample into the flow cell. In one embodiment, the sensor 10, 1 00 can provide responses by using the SERS spectroscopy or the UV-Vis spectrometer, which shows the light modulation after passing the surface plasmon resonance (SPR) happening at the chip surface. When a coating is disposed on the sensor chip having an affinity for a target analyte in a sample, the response with the target analyte may be compared to a control sample without the target analyte.
In accordance with yet another aspect of the present invention, there is provided a method for determining a presence of an analyte in a sample in a surface plasmon resonance system. The method comprises introducing an energy source 202 to excite surface plasmon resonance and flowing an analyte over a flow cell 204 comprising a sensor 10, 100 as described having a plurality of shaped protuberances 12, 1 02. In addition, the method comprises combining signals from one or more detectors 208 to detect a change in proximity of the sensor 10, 100. The shaped protuberances 12, 1 02 may be sized and from any suitable material as described herein. In one embodiment, the shaped protuberances 12, 102 are functionalized with one or more agents that have an affinity for a target analyte. Exemplary agents include, but are not limited to proteins, oligonucleotides, DNA molecules, RNA molecules, lipids, carbohydrates, glycoproteins, lipoproteins, peptides, polysaccharides, polypeptides or chemical entities.
In one embodiment, the one or more detectors 208 receive electromagnetic radiation transmitted, scattered, absorbed or reflected by the sensor 10, 100, and sense spectrum and image changes. The spectrum and image changes may be reflected in changes in frequency, intensity, phase, or other characteristic of opto-electrical signals. In a particular embodiment, the one or more detectors 208 may include surface enhanced Raman scattering (SERS) spectroscopy, spectrometer, and image detectors.
In accordance with yet another aspect of the present invention, there is provided a method for making a plasmon sensor. The method comprises depositing a polymer material in a first substrate having a plurality of tapered recesses etched therein. In addition, the method comprises removing the polymer material from the first substrate using a second substrate to provide a plurality of tapered protuberances on a surface of the second substrate. Further, the method includes depositing a layer of a metallic material, e.g., metallic coating 20, over the plurality of tapered protuberances to form the sensor.
In one embodiment, the first substrate may be generated by a self-assembly method which comprises depositing, e.g., by spin-coating, silica particles and polymer on the surface of the first substrate, etching polymer and depositing a mask layer of chromium which produce a plurality of periodic nanohole arrays, and lastly, aniotropic wet etching through the nanohole arrays to provide the tapered recesses. The mask layer may be of a metallic material, e.g., chromium, which is deposited by electron- beam evaporation or any other suitable method.
The shaped protuberances described herein may be sized, shaped, and spaced apart as described above, such as in the form of spaced apart nanopyramids or nanodimples having at least one dimension between 1 -1000 nm. It is appreciated that the dimensions of the shaped protuberance can be adjusted by changing the size of the templating silica spheres and the etching conditions, such as temperature, duration, and etchant concentration.
In accordance with another aspect of the present invention, there is provided a method for making a surface plasmon sensor. The method comprises preparing a colloidal suspension comprising silica and a polymer. In addition, the method comprises spin coating the colloidal suspension to form crystal colloids in the polymer and etching to selectively remove polymer to provide an array of dimpled protuberances comprising the crystal colloids. Thereafter, the method includes depositing a metallic layer over the dimpled protuberances to form the sensor. In one embodiment, the etching is done from 15 to 1 05 seconds effective to provide the plurality of dimpled protuberances. In a particular embodiment, the etching is done from 90 to 105 seconds.
The applications of SPR sensor for biosensing incorporate a biorecognition coating that interacts with the molecules in samples. While the molecules are absorbed, deabsorbed, cleaved, or modified on the surface, the change of the dielectric layer on the surface will cause modulation of sensor output. Aspects of the present invention may be used for analyzing kinetic and thermodynamic interaction of biomolecules including protein, peptide, oligonucleotide, etc. In addition, aspects of the present invention may be used for any other chemical or physical sensing while the transducing medium is altered by analytes or reacts with analytes.
EXAMPLE 1
A colloidal suspension was prepared as followed. Uniform silica particles were synthesized with a diameter of 300 nm by the well-known Stober method. The synthesized silica particles are purified in 200-proof ethanol by multiple (at least 3) centrifugation/redispersion cycles. After removing the supernatant solution, centrifuged silica particles were dispersed in an ethoxylated trimethylolpropane triacrylate monomer (ETPTA monomer) to make silica/ETPTA suspensions with 20% particle volume fraction. The resulting suspensions were filtered using syringe filters with 5-micron pores. 1 wt% of photoinitiator (Darocur 1 173) was then added and the resulting suspension was stored in an open glass vial in dark for one day to let any remaining ethanol to evaporate. A transparent suspension was obtained.
Spin Coating of Colloidal Suspensions: The above colloidal suspension was dispensed on a 4-inch silicon wafer and is spread uniformly on the wafer. Spin-coating of the suspension was performed on a programmable spin coater. The spinning process used is as follows: 200 rpm for 2 min, 300 rpm for 2 min, 1000 rpm for 1 min, 3000 rpm for 20 s, and 6000 rpm for 1 .5 min. Spin coating resulted in a distinctive hexagonal pattern on the silicon wafer. UV exposure of the spin-coated sample for 4 s allowed the monomer to be polymerized to get a colloidal crystal/polymer nanocomposite.
Fabrication of Nanodimples by Reactive Ion Etching(RIE): The spin-coated nanocomposite was oxygen-RIE-etched using the following conditions: 40 mTorr oxygen pressure, 40 seem flow rate and 100 W power for 120 s. The dry etched sample was then dipped in a 2 wt% hydrofluoric acid aqueous solution to remove the exposed silica particles. The resulting sample was then rinsed with Dl water and blow dried with nitrogen. A 5 nm chromium followed by 100 nm gold were then sputtered on the sample.
The UV exposure was performed on a Xenon Pulsed UV curing system RC 742. RIE was carried out on an Unaxis Shutterlock RIE/ICP reactive ion etcher. Metallization was performed using a Kurt Lesker CMS-1 8 Multi Target Sputter Deposition System. A Laurell spin coater was used to conduct the spin coating experiment.
EXAMPLE 2
The SERS performance of the templated nanodimple arrays was evaluated using benzenethiol as a model compound because of its excellent affinity to gold, as well as its large Raman scattering cross-section. FIG. 4 shows the SER spectra obtained at the templated nanodimple arrays prepared by different durations of oxygen plasma etching. These spectra were taken using a 785 nm diode laser at 2.5 mW with an integration time of 10 s. The 75 and 90 s etched samples exhibit the most distinctive SERS peaks whose positions and relative amplitude match with those in the literature for benzenethiol molecules adsorbed on Au nanoparticles and structured Au surfaces. The SERS enhancement factor for these samples is estimated to be more than 10 7 , which compares favorably to that obtained for nanofabricated plasmonic structures by using expensive nanolithographic technologies.
It is well-known that the surface plasmon resonances enabled by plasmonic nanostructures play an important role in determining the amplitude of SERS enhancement. The greatest enhancement occurs when surface plasmon resonances are present at both the laser excitation wavelength and the Raman scattered wavelength. To evaluate the surface plasmon resonance of the templated Au nanodimple arrays, the present inventors measured optical reflection at normal incidence. FIG. 5 shows the reflectance spectra obtained for the Au nanodimple samples with different oxygen plasma etching durations. The position of the laser excitation wavelength, 785 nm, is also indicated by the dashed line. The 75 and 90 s etched samples exhibit strongest absorption at the laser excitation wavelength, corresponding well to the strongest SER enhancement as observed in FIG. 4.
Ultrasensitive SPR biosensors may be particularly useful because they can offer a direct and label-free platform for rapid screening tests. As shown in FIG. 5, the templated nanodimple arrays show strong and tunable SPR adsorption by simply controlling the plasma etching duration. The present inventors therefore used the templated nanodimples as SPR sensors. A sandwich cell, consisting of a gold nanodimple array, a 2 mm thick polydimethylsiloxane (Sylgard 184) spacer, and a glass microslide, was used to evaluate the reflection from the nanodimples when the cell was filled with glycerol solutions of different refractive indices. A high-resolution spectrometer (HR4000, Ocean Optics) with a tungsten halogen light source (LS-1 ) and a reflection probe (R600-7) was used for the optical measurements. The angle of incidence was controlled at 45° by using a RPH-1 probe holder. FIGS. 6-8 show the reflection spectra obtained from glycerol solutions of different refractive indices on 30, 60, and 1 20 s etched nanodimple samples. A red-shift of the maximum reflection wavelength is observed as the solution refractive index increases. The sensitivity of these nanodimple arrays was evaluated to be 416, 477, and 520 nm per refractive index unit (nm/RIU).
EXAMPLE 3 To create gold nanopyramid arrays, spin-coating was first used to generate non- close-packed (NCP) colloidal mono layers of hexagonally ordered silica particles on a (1 00) silicon wafer. The ordered silica particles were then used as shadow masks during electron-beam evaporation of chromium to create periodic nanohole arrays. Anisotropic wet etching was then utilized through the metal nanohole arrays to make inverted pyramidal pits in silicon. Wafer-scale gold nanopyramid arrays were finally templated by sputtering 500 nm of gold on the silicon mold, followed by a simple adhesive peeling process with a glass microslide as the substrate. The template stripping produces ultra-smooth pure metal films with surface-plasmon-propagation lengths approaching theoretical values for perfectly flat films. Images of the produced gold nanopyramids are again shown in FIGS.1 A-1 B.
A sandwich cell, consisting of a gold nanopyramid array, a 2 mm thick polydimethylsiloxane (PDMS, Sylgard 1 84) spacer, and a bare glass microslide, was used to evaluate the optical reflection from the nanopyramids when the cell was filled with glycerol solutions with different refractive indices. The glycerol solution (Sigma- Aldrich) was injected by a 250 μιη I.D. needle. The refractive indices of glycerol solutions were measured by a refractometer. Prior to each measurement, glycerol solution was injected and extracted several times to remove residual glycerol solution from the last run. A tungsten halogen light source (LS-I, Ocean Optics) and a reflection probe (R600-7, Ocean Optics) were used for the optical measurements. The angle of incidence on the sandwich cell was controlled at 45° by using a reflection probe holder (RPH-I, Ocean Optics). The reflected light was collected by a high- resolution spectrometer (HR4000, Ocean Optics). FIG. 9 shows the normalized reflection spectra obtained from glycerol solutions of different refractive indices. A red-shift of the maximum reflection wavelength is observed as the solution refractive index increases. The sensitivity of the nanopyramid array was evaluated to be 239 nm per refractive index unit (nm/RIU) (see FIG. 1 1 ) and is favorably comparable to other grating coupler-based SPR sensors. EXAMPLE 4
Experimental measurements were complemented by theoretical simulation using a FDTD model under a Lumerical Solutions platform. The dimensions of the gold nanopyramid were obtained from the SEM image in FIG. 1 (b). The base length, nanopyramid height, lattice spacing between adjacent nanopyramids, and tip radius of curvature were set as 300 nm, 200 nm, 420 nm, and 50 nm, respectively. Bloch boundary conditions were used in both x- and y-axis because of the periodic structure of nanopyramids. The metal boundary condition was set at the bottom for providing a radiation boundary and the perfect matched layers (PML) approach was used at the top for providing an absorption boundary condition so that little or no electromagnetic radiation is reflected back into the simulation region. Plane waves with wavelength from 500 to 700 nm were used for the modeling. To simplify the simulation, the real incident angle at a gold nanopyramid array was calculated using Snell's law by considering the incident angle in air (45 5 to the normal incidence) and the refractive indices of air and liquid as: η 3 , Γ x sin(6 a ir) = η 9 ι 355 x sin(e g i aS s) = η Ν ί x sin(0ii quic i), where n is the refractive index and Θ is the angle of incidence. For water (η 3 , Γ = 1 , Q a \ r = 45 5 , and niiquid = 1 -33), Giiquid is calculated to be 32.1 5 and the same rule is applied to testing all glycerol solutions with different refractive indices. On the 0 th order signals were simulated due to the narrow acceptance angle (24.8 in air) of the reflection probe.
FIG. 10(a) shows FDTD-simulated reflection spectra from a gold nanopyramid array immersed in glycerol solutions of different refractive indices. A red-shift of the maximum reflection is observed as the refractive index increases, showing the same trend as the experimental result in FIG. 9. FIG. 1 0(b) shows a comparison between experimental and theoretical reflection from gold nanopyramid arrays with different tip sharpness. The peak position of the experimental spectrum is closer to that of the simulation with 10 nm tips while the shape of the spectrum is more similar to that of the simulation with 50 nm tips. The difference in the maximum reflection may result from the discrepancy between the real and ideal arrays of gold nanopyramids, such as defects (e.g., grain boundaries) and the sharpness of edges and tips. The apparent broadening of the peak could be caused by the defects in the templated nanopyramid array. It is well-known that structural defects such as polycrystalline domains and point and line defects can significantly broaden optical spectra.
The maximum reflection wavelength of both simulated and experimental spectra obtained from solutions of different refractive indices is summarized in FIG. 4. It is apparent that the simulated reflection peaks are red-shifted by -20 to 30 nm than the experimental results. The sensitivity of the simulated reflection is calculated to be 314 nm/RIU, larger than that of the experimental value (239 nm/RIU). The difference in sensitivity could also result from the defects and the nonideal sharpness of tips and edges of the nanopyramid arrays.
EXAMPLE 5
The biosensing performance of the templated gold nanopyramid arrays was investigated by using Rabbit IgG antibody to alcohol dehydrogenase, an enzyme that catalyzes the oxidation of alcohol. First, the gold nanopyramid array was treated with Protein A, a protein that resides in the microbial wall of Staphylococcus aureus, to enhance the conjugation of rabbit IgG on gold nanopyramids. Lyophilized
Protein A (MP Biomedical) was reconstituted to 50 μg/μL with phosphate buffer saline (PBS) containing 0.1 37M NaCI at pH 7.4 (Fisher) and stored at -20°C. Prior to use, the stock solution of Protein A was first diluted to 5 μg/μL, then put on a clean gold nanopyramid array sample and subsequently incubated overnight at 4°C. After incubation, the gold nanopyramid array was rinsed three times with PBS buffer to wash away un-adsorbed Protein A and incubated with 10 μg/μL of rabbit polyclonal to alcohol dehydrogenase (abeam) overnight at 4°C. The corresponding normalized reflection spectrum due to the adsorption of anti-alcohol dehydrogenase is shown in FIG. 12 with a peak wavelength of 595.9 nm (black line). 40 ng/mL of alcohol dehydrogenase (MP Biomedicals) in PBS was then added on the array and incubated for 1 hr at room temperature, followed by three times rinsing with PBS buffer for removing unbound residue. A red shift of peak wavelength from 595.9 nm to 602.3 nm (red line) was observed when alcohol dehydrogenase was added to the anti-alcohol dehydrogenase-modified nanopyramid sample. The detection selectivity was also tested by using casein, a protein commonly used as a blocking reagent. After the conjugation of alcohol dehydrogenase, the same array was exposed to 1 % casein in PBS (Thermo). As expected, no obvious peak shift (blue line) was noticed due to the negligible binding of nonspecific protein casein on the sample.
While various embodiments of the present invention have been shown and described herein, it will be obvious that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein. Accordingly, it is intended that the invention be limited only by the spirit and scope of the appended claims. The teachings of any reference cited herein are incorporated herein in their entirety to the extent not inconsistent with the teachings herein.