Abstract:
Packaged NERS-active structures are disclosed that include a NERS substrate having a NERS-active structure thereon, and a packaging substrate over the NERS substrate having an opening therethrough, the opening in alignment with the NERS-active structure. A membrane may cover the opening in the packaging substrate. In order to perform nanoenhanced Raman spectroscopy, the membrane may be removed, and an analyte placed on the NERS substrate adjacent the NERS-active structure. The membrane may be replaced with another membrane after the analyte has been placed on the substrate. The membrane may maintain the pristine state of the substrate before it is deployed, and the replacement membrane may preserve the substrate and analyte for archival purposes. Also disclosed are methods for performing NERS with packaged NERS-active structures.

Description:
FIELD OF THE INVENTION 
     The invention relates to nanoenhanced Raman scattering (NERS). More particularly, the invention relates to packaging for NERS-active structures, protection for surfaces of NERS-active structures, also including methods for forming packaging for NERS-active structures, and methods for packaging NERS-active structures. 
     BACKGROUND OF THE INVENTION 
     Raman spectroscopy is a well-known technique for performing chemical analysis. In conventional Raman spectroscopy, high intensity monochromatic light provided by a light source, such as a laser, is directed onto an analyte (or sample) that is to be chemically analyzed. A majority of the incident photons are elastically scattered by the analyte molecule. In other words, the scattered photons have the same energy, and thus the same frequency, as the photons that were incident on the analyte. However, a small fraction of the photons (i.e., about 1 in 10 7  photons) are inelastically scattered by the analyte molecules. These inelastically scattered photons have a different frequency than the incident photons. This inelastic scattering of photons is termed the “Raman effect.” The inelastically scattered photons may have frequencies greater than or, more typically, less than the frequency of the incident photons. 
     When an incident photon collides with a molecule, energy may be transferred from the photon to the molecule or from the molecule to the photon. When energy is transferred from the photon to the molecule, the scattered photon will emerge from the sample having a lower energy and a corresponding lower frequency. These lower-energy Raman scattered photons are commonly referred to in Raman spectroscopy as the “Stokes radiation.” A small fraction of the analyte molecules are already in an energetically excited state. When an incident photon collides with an excited molecule, energy may be transferred from the molecule to the photon, which will emerge from the sample having a higher energy and a corresponding higher frequency. These higher-energy Raman scattered photons are commonly referred to in Raman spectroscopy as the “anti-Stokes radiation.” 
     The Stokes and the anti-Stokes radiation is detected by a detector, such as a photomultiplier or a wavelength-dispersive spectrometer, which coverts the energy of the impinging photons into an electrical signal. The characteristics of the electrical signal are at least partially a function of the energy (or wavelength, frequency, wave number, etc.) of the impinging photons and the number of the impinging photons (intensity). The electrical signal generated by the detector can be used to produce a spectral graph of intensity as a function of frequency for the detected Raman signal (i.e., the Stokes and anti-Stokes radiation). A unique Raman spectrum corresponding to the particular analyte may be obtained by plotting the intensity of the inelastically scattered Raman photons against the frequency thereof. This unique Raman spectrum may be used for many purposes such as identifying an analyte, identifying chemical states or bonding of atoms and molecules in the analyte, and determining physical and chemical properties of the analyte. Raman spectroscopy may be used to analyze a single molecular species or mixtures of different molecular species. Furthermore, Raman spectroscopy may be performed on a number of different types of molecular configurations, such as organic and inorganic molecules in either crystalline or amorphous states. 
     Molecular Raman scattering of photons is a weak process. As a result, powerful, costly laser sources typically are used to generate high intensity excitation radiation to increase the otherwise weak Raman signal for detection. Nanoenhanced Raman scattering (NERS) is a technique that allows for generation of a stronger Raman signal from an analyte relative to conventional Raman spectroscopy. In NERS, the analyte molecules are adsorbed onto, or placed adjacent to, an active metal surface or structure (an “NERS-active structure”). The interactions between the molecules and the active structure cause an increase in the strength of the Raman signal. The mechanism of Raman signal enhancement exhibited in NERS is not completely understood. Two main theories of enhancement mechanisms have been presented in the literature: electromagnetic enhancement and chemical (or “first layer”) enhancement. (For further discussion of these surface enhancement mechanism theories, see A. M. Michaels, M. Nirmal, &amp; L. E. Brus, “Surface Enhanced Raman Spectroscopy of Individual Rhodamine 6G Molecules on Large Ag Nanocrystals,”  J. Am. Chem. Soc.  121, 9932-39 (1999)). 
     Several NERS-active structures have been employed in NERS techniques, including activated electrodes in electrolytic cells, activated metal colloid solutions, and activated metal substrates such as a roughened metal surface or metal “islands” formed on a substrate. For example, it has been shown that adsorbing analyte molecules onto or near a specially roughened metal surface made from gold or silver may enhance the effective Raman scattering intensity by factors of between  103  and  106  when averaged over the illuminated area of the sample. 
     The NERS substrate may be easily contaminated. Maintaining the pristine state of the substrate before it is deployed may be difficult. One solution is a disposable NERS substrate; however, this may be expensive. Additionally, it may be desirable to preserve a NERS substrate for archival purposes, such that analyte molecules may be retested. This may be useful, for example, to serve as evidence of the presence of materials used in a weapon. 
     Accordingly, there is a need for a protection method for NERS-active structures, both before and after use. Packaging may provide the needed protection. Thus, there is a need for packaging for NERS-active structures, methods for forming packaging for NERS-active structures, and methods for packaging NERS-active structures. 
     BRIEF SUMMARY OF THE INVENTION 
     A packaged NERS-active structure is disclosed that includes a substrate, at least one NERS-active structure disposed on the substrate, a packaging substrate having at least one opening therethrough disposed on the substrate, the opening being aligned with the NERS-active structure, and a removable membrane covering the opening. 
     A method of packaging a NERS active structure is disclosed that includes providing at least one NERS active structure on a first substrate, attaching a second substrate having an opening therethrough on the first substrate, the opening providing access to the NERS active structure, and providing a membrane covering the opening in the second substrate. 
     A method of preserving an analyte on a NERS substrate for archiving is disclosed that includes providing a packaged NERS-active structure comprising: a substrate; at least one NERS-active structure disposed on the substrate; a packaging substrate having at least one opening therethrough disposed on the substrate, the opening being aligned with the NERS-active structure; and a membrane covering the opening. The membrane covering the opening may be removed, an analyte molecule may be placed adjacent the at least one NERS-active structure, and the opening may be covered. 
     A method for forming a packaged NERS-active structure is disclosed that includes providing a substrate having a surface, affixing at least one NERS-active structure on the surface of the substrate, adhering a packaging substrate to the surface of the substrate, the packaging substrate having at least one opening therethrough, the at least one opening proving access to the NERS-active structure, and covering the at least one opening with a removable membrane. 
     The features, advantages, and alternative aspects of the present invention will be apparent to those skilled in the art from a consideration of the following detailed description taken in combination with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
       While the specification concludes with claims particularly pointing out and distinctly claiming that which is regarded as the present invention, the advantages of this invention can be more readily ascertained from the following description of the invention when read in conjunction with the accompanying drawings in which: 
         FIG. 1A  is an exploded view of a first embodiment of a packaged NERS-active structure according to the invention; 
         FIG. 1B  is an assembled view of the packaged NERS-active structure of  FIG. 1A ; 
         FIG. 2  is an exploded view of a second embodiment of a packaged NERS-active structure according to the invention; and 
         FIG. 3  is a schematic diagram of an exemplary system for performing nano-enhanced Raman spectroscopy using the packaged NERS-active structures of  FIGS. 1A ,  1 B, and  2 . 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention, in a number of embodiments, includes packaging for NERS-active structures, protection for surfaces of NERS-active structures, methods for forming packaging for NERS-active structures, and methods for packaging NERS-active structures. 
     The term “NERS-active structure” as used herein means a structure that is capable of increasing the number of Raman-scattered photons that are scattered by a molecule when the molecule is located adjacent to the structure, and the molecule and structure are subjected to electromagnetic radiation. 
     The term “NERS-active material” as used herein means a material that, when formed into appropriate geometries or configurations, is capable of increasing the number of Raman-scattered photons that are scattered by a molecule when the molecule is located adjacent the material, and the molecule and material are subjected to electromagnetic radiation. NERS-active materials can be used to form a NERS-active structure. 
     The term “analyte molecule” as used herein means a molecule upon which it is desired to perform NERS. 
     It should be understood that the illustrations presented herein are not meant to be actual views of any particular NERS-active structure, but are merely idealized representations which are employed to describe the present invention. Additionally, for ease of discussion, elements common to  FIGS. 1 through 3  retain the same numerical designation. 
     A first embodiment of a packaged NERS-active structure  10  according to the invention is shown in  FIGS. 1A and 1B .  FIG. 1A  depicts the packaged NERS-active structure  10  in an exploded view, and  FIG. 1B  illustrates the packaged NERS-active structure  10  in assembled form. The packaged NERS-active structure  10  includes a NERS-active substrate  100  and a packaging substrate  200 . The NERS-active substrate may comprise, by way of example, one of silicon, glass, quartz or plastic material. The NERS-active substrate  100  may include at least one NERS-active structure  120  on a first surface  110  thereof.  FIG. 1A  depicts a plurality of NERS-active structures  120  disposed on the first surface  110  in an array. Optionally, the plurality of NERS-active structures  120  may be randomly positioned. The at least one NERS-active structure  120  may be on a central region  130  of the substrate first surface  110 . The NERS-active structures  120  may be formed of a NERS-active material, such as, for example gold, silver, copper, platinum, palladium, aluminum, or any other material that will enhance the Raman scattering of photons by analyte molecules positioned adjacent thereto. 
     The packaging substrate  200  may include an opening  220  therethrough, as depicted in  FIG. 1B . The opening  220  may be sized to match the central region of the substrate first surface  110 , enabling the NERS-active structures  120  to be accessed through the opening  220 . A membrane  210  may cover the opening  220 , as illustrated in  FIG. 1A . The membrane  210  may be peeled back, as depicted in  FIG. 1B , or otherwise removed to expose the NERS-active structures  120 . The packaging substrate  200  may comprise one of silicon, glass, quartz, or plastic material. 
     One example of a suitable membrane  210  is a thin metal film. A current may be used to burn the metal film off to expose the NERS-active structures below. Thermally or optically degradable polymer films may also be used. Methods of removing a degradable polymer film include, by way of example and not limitation, laser ablation, microwave or acoustic decomposition, electrical, or thermal burn-off. A degradable polymer film covering the area of a NERS-site, which may be between about 1 μm 2  and about 100 μm 2  may be removed with a laser having a power of between about 2 and about 6 mW. 
     The membrane  210  may be reusable. A polymer film may be peeled back, for example, by using robotics, before an analyte is disposed on the substrate  100 . The packaged NERS-active structure  10  may be used to perform NERS, as described below, then robotics may be used to replace the membrane  210 , the sealing the NERS-active structure  120  and adjacent analyte (not shown) for archiving. The packaged NERS-active structure  10  may be stored, and the analyte may be tested again in the future. 
     A surface  215  of the membrane  210  (see  FIG. 1B ) may be passivated or coated with an inert substance such as fluorinated hydrocarbons. The coated surface  215  may be the surface facing the NERS-active structure  120 , which protects the central region  130  of the substrate first surface  110  and the NERS-active structure  120  from unnecessary contamination. 
     The packaging substrate  200  may be secured to the substrate  100 , for example, with a bonding material. One example of a suitable bonding material is a two-component reactive adhesive. Sealants and resins including acrylic, anaerobic materials, conductives, epoxy, polysulfides, polyurethanes, UV curable and other polymers may also be suitable. The packaging substrate  200  may be secured to the periphery of the substrate first surface  110 , and the central region  130  may remain accessible through the opening  220  of the packaging substrate  200 . 
       FIG. 2  depicts a second embodiment of a packaged NERS substrate of the present invention. A NERS coupon  300  may be formed of a substrate made of, for example, silicon, glass, quartz, or plastic, with an array of NERS sites  330  thereon. The NERS coupon  300  may be between about 1 cm 2  to about 10 cm 2  Each NERS site  330  may be between about 1 μm 2  to about 200 μm 2 . Anywhere from one to several millions of NERS sites  330  may be disposed on the NERS coupon  300 . Each NERS site  330  comprises at least one NERS-active structure  120 , as shown in  FIG. 1A . 
     A packaging substrate  400  may include a plurality of openings  420  therein. Each opening  420  may be covered with a membrane  410 . The packaging substrate may include a first surface  430  and an opposing, second surface  440 . The second surface may be adjacent to the NERS coupon  300 . The membranes  410  may be disposed on the first surface  430 . Each opening  420  may optionally be tapered, with the area of the opening  424  at the first surface  430  being less than the area of the opening  426  at the second surface  440 . The area of the membrane  410  may be greater than the area of the opening  424  at the first surface  430 , enabling the membrane  410  to be adhered to, and supported by, the first surface  430  of the packaging substrate  400 . 
     The openings  420  and associated membranes  410  may be formed using conventional microengineering techniques. For example, the packaging substrate  400  may be coated with a mask material on the first surface  430  and the second surface  440 . The coating of the mask material on the second surface  440  may be patterned according to the desired locations of the openings  420 . The packaging substrate  400  may be etched from the second surface  440  to form the openings  420 . Each membrane  410  may be deposited in the desired location over the mask material on the first surface  430 . For example, a negative photoresist may be used to define the desired locations of each membrane  410 , and a layer comprising gold may be deposited by evaporation. The membranes  410  may be defined using a lift-off procedure, that is, by removing the resist and overlying portions of the gold layer in the undesirable locations, leaving the portions of the gold layer in the form of membranes  410 . 
     Optionally, conductive traces  450  may be provided on the first surface  430  of the packaging substrate  400 . The conductive traces  450  may be in electrical communication with each membrane  410  and may be used to burn off a conductive membrane, such as, for example, a metal film. Each membrane  410  may be removed selectively, with the other membranes  410  remaining intact. Alternatively, all of the membranes  410  may be removed simultaneously. 
     Thermally or optically degradable polymer films may also be used as the membranes  410 . Methods of removing a degradable polymer film include, by way of example and not limitation, laser ablation, microwave or acoustic decomposition, electrical burn-off, or thermal burn-off. A degradable polymer film covering the area of a NERS-site, which may be between about 1 μm 2  and about 100 μm 2 , may be removed with a laser having a power between about 2 and about 6 mW. 
     The membrane  410  may be reusable. A polymer film may be peeled back, for example, by using robotics, before an analyte is disposed on a NERS site  330  of the NERS coupon  300 . The packaged NERS-active structure  20  may be used to perform NERS at one NERS site  330  as described hereinbelow, then robotics may be used to replace the membrane  410 , sealing the NERS-active structure  120  and adjacent analyte (not shown) for archiving. The packaged NERS-active structure  20  may be stored, or other NERS sites  330  may be used for analyte testing. In this fashion, any analyte sealed within the packaged NERS-active structure  20  may be tested again in the future. 
     An exemplary NERS system  500  according to the invention is illustrated schematically in  FIG. 3 . The system  500  may include one of the exemplary packaged NERS-active structures  10 ,  20  and may be used to perform nano-enhanced Raman spectroscopy. The NERS system  500  may include a sample or analyte stage  510 , an excitation radiation source  520 , and a detector  530 . The analyte stage  510  may include one of the packaged NERS-active structures  10 ,  20 . The NERS system  500  also may include various optical components  540  positioned between the excitation radiation source  520  and the analyte stage  510 , and various optical components  550  positioned between the analyte stage  510  and the detector  530 . 
     The excitation radiation source  520  may include any suitable source for emitting radiation at the desired wavelength, and may be capable of emitting a tunable wavelength of radiation. For example, commercially available semiconductor lasers, helium-neon lasers, carbon dioxide lasers, light emitting diodes, incandescent lamps, and many other known radiation-emitting sources may be used as the excitation radiation source  520 . The wavelengths that are emitted by the excitation radiation source  520  may include any suitable wavelength for properly analyzing the analyte using NERS. An exemplary range of wavelengths that may be emitted by the excitation radiation source  520  includes wavelengths between about 350 nm and about 1000 nm. 
     The excitation radiation emitted by the source  520  may be delivered either directly from the source  520  to the analyte stage  510  and the packaged NERS-active structure  10 ,  20 . Alternatively, collimation, filtration, and subsequent focusing of the excitation radiation may be performed by optical components  540  before the excitation radiation impinges on the analyte stage  510  and the packaged NERS-active structure  10 ,  20 . 
     The packaged NERS-active structure  10 ,  20  of the analyte stage  510  may enhance the Raman signal of the analyte, as previously discussed. In other words, irradiation of the NERS-active structure  10 ,  20  by excitation radiation may increase the number of photons inelastically scattered by an analyte molecule positioned near or adjacent to the packaged NERS-active structure  10 ,  20 . 
     The Raman scattered photons may be collimated, filtered, or focused with optical components  550 . For example, a filter or a plurality of filters may be employed, either as part of the structure of the detector  530  or as a separate unit that is configured to filter the wavelength of the excitation radiation, thus allowing only the Raman scattered photons to be received by the detector  530 . 
     The detector  530  receives and detects the Raman scattered photons and may include a monochromator (or any other suitable device for determining the wavelength of the Raman scattered photons) and a device such as, for example, a photomultiplier for determining the quantity of Raman scattered photons (intensity). 
     Ideally, the Raman scattered photons are scaffered isotropically, being scattered in all directions relative to the analyte stage  510 . Thus, the position of the detector  530  relative to the analyte stage  510  is not particularly important. However, the detector  530  may be positioned at, for example, an angle of 90° relative to the direction of the incident excitation radiation to minimize the intensity of the excitation radiation that may be incident on the detector  530 . 
     To perform NERS using the system  500 , a user may remove the membrane  210  and provide an analyte molecule or molecules adjacent to the NERS-active structure  120  of the packaged NERS-active structure  10 ,  20 . The analyte and the NERS-active structure  120  are irradiated with excitation radiation or light from the source  520 . Raman scattered photons scattered by the analyte are then detected by the detector  530 . The membrane  210  may be replaced, or a new membrane  210  may be provided to replace the membrane  210 , and preserve the analyte molecule or molecules within the packaged NERS-active structure. 
     The structures and systems disclosed herein may also be used to perform enhanced hyper-Raman spectroscopy. When excitation radiation impinges on an analyte molecule, a very small number of photons may be scattered at frequencies corresponding to the higher order harmonics of the excitation radiation, such as the second and third harmonics (i.e., twice or three times the frequency of the excitation radiation). Some of these photons may have a frequency that is Raman-shifted relative to the frequencies corresponding to the higher order harmonics of the excitation radiation. These higher order Raman-scattered photons can provide information about the analyte molecule that cannot be obtained by first order Raman spectroscopy. Hyper-Raman spectroscopy involves the collection and analysis of these higher order Raman-scattered photons. 
     Although the foregoing description contains many specifics, these are not to be construed as limiting the scope of the present invention, but merely as providing certain exemplary embodiments. Similarly, other embodiments of the invention may be devised which do not depart from the spirit or scope of the present invention. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents, rather than by the foregoing description. All additions, deletions, and modifications to the invention, as disclosed herein, which fall within the meaning and scope of the claims, are encompassed by the present invention.