Abstract:
A Raman-active particle as well as a method of making a Raman-active particle are described. The Raman-active particle includes a core particle and a coating. The coating substantially covers the core particle. A Raman-active analyte is at least partially within the coating. The method of making a Raman-active particle includes i) providing a colloidal solution comprising a core particle; ii) providing a coating or coating precursor to the colloidal solution to form a resulting solution; and iii) providing a Raman-active analyte to the resulting solution. A method of conducting an assay is also described. The method includes: i) attaching a Raman-active particle to a targeted moiety; ii) measuring the Raman spectrum of the Raman-active particle; and iii) correlating the Raman spectrum to the presence of the targeted moiety.

Description:
BACKGROUND OF THE INVENTION 
     The present invention generally relates to Raman-active particles and methods of making and using the same, and more particularly relates to Raman-active particles with an analyte at least partially within a coating of the Raman-active particle and methods of making the same. 
     DESCRIPTION OF RELATED ART 
     In some known technologies, a Raman-active analyte is incorporated into a material, such as the Raman-active particle referred to in U.S. Pat. No. 6,514,767. Some Raman-active particles, as in U.S. Pat. No. 6,514,767, typically have a core particle (also referred to as a core), a Raman-active analyte, and a coating, (also referred to as an encapsulant). Typically, a linker is provided which allows the deposition of a coating material onto the core particle. The coating inhibits Raman-active particles from aggregating. A conventional Raman-active particle  10  is illustrated in  FIG. 1  as including a coating  14  surrounding a core particle  12  and an analyte  16 . 
     Known methods of making Raman-active particles  10  involve incorporating a Raman-active analyte into the Raman-active particles  10  before a coating solution is added. A disadvantage with adding the analyte  16  before initiating the coating  14  is uncontrolled aggregation of core particles  12 . Aggregation occurs because the analyte  16 , which causes core aggregation and flocculation, binds more strongly to the surface of the core particle  12  than the linker, thereby displacing the linker. The analyte  16  binds more strongly to the core particle than the linker even before the coating  14  begins to form and colloid flocculation can occur. In some known methods, the amount of analyte  16  added is equal to the amount of linker added, enhancing the likelihood that colloid flocculation will occur, since the analyte  16  could theoretically completely displace the linker. 
     Thus, an improved method of making Raman-active particles that addresses some of the deficiencies exhibited by known methods is still needed. Also needed are Raman-active particles that address some of the deficiencies exhibited in known Raman-active particles. 
     SUMMARY 
     The purpose and advantages of embodiments of the invention will be set forth and apparent from the description of exemplary embodiments that follows, as well as will be learned by practice of the embodiments of the invention. Additional advantages will be realized and attained by the methods and systems particularly pointed out in the written description and claims hereof, as well as from the appended drawings. 
     An embodiment of the invention provides a Raman-active particle. The Raman-active particle includes a core particle and a coating. The coating substantially covers the core particle and a Raman-active analyte is at least partially within the coating. 
     A second embodiment provides a method of making a Raman-active particle. The method includes: i) providing a colloidal solution comprising a core particle and ii) providing a coating or coating precursor to the colloidal solution to form a resulting solution; and iii) providing a Raman-active analyte to the resulting solution. 
     A third embodiment provides a method of conducting an assay. The method includes: i) attaching a Raman-active particle to a targeted moiety; ii) measuring a Raman spectrum of the Raman-active particle; and iii) correlating the Raman spectrum to the presence of the targeted moiety. The Raman-active particle includes: a core particle and a coating. The coating substantially covers the core particle and a Raman-active analyte is at least partially within the coating. 
     A fourth embodiment provides a Raman-active particle. The Raman-active particle includes a core particle with a metallic surface and a coating. The coating substantially covers the core particle and includes silica. A Raman-active analyte is at least partially within the coating. 
     A fifth embodiment provides a method of making a Raman-active particle. The method includes: i) providing a colloidal solution comprising a core particle; ii) providing a coating or coating precursor to the colloidal solution to form a resulting solution; and iii) providing a Raman-active analyte to the resulting solution. The core particle has a metallic surface and the coating includes silica. 
     The accompanying figures, which are incorporated in and constitute part of this specification, are included to illustrate and provide a further understanding of the method and system of the invention. Together with the description, the drawings serve to explain the principles of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic representation of a known Raman-active particle; 
         FIG. 2  is a schematic representation of a Raman-active particle wherein the analyte is dispersed within the coating in accordance with an embodiment of the invention; 
         FIG. 3  is a schematic representation of a Raman-active particle wherein the analyte partially surrounds a coated Raman-active particle in accordance with an embodiment of the invention; 
         FIG. 4  is a schematic representation of a Raman-active particle wherein the analyte is embedded within the coating in accordance with an embodiment of the invention; 
         FIG. 5  is a schematic representation of a Raman-active particle with a plurality of core particles in accordance with an embodiment of the invention; 
         FIG. 6  is a schematic representation of a Raman-active particle with a plurality of core particles in accordance with an embodiment of the invention; 
         FIG. 7  is a schematic representation of a method of making a Raman-active particle in accordance with an embodiment of the invention; 
         FIG. 8  is a flow chart of a method of making a Raman-active particle in accordance with an embodiment of the invention; 
         FIG. 9  are Raman spectra of an embodiment of Raman-active particles with 4-Mercaptobenzoic acid (MBA) and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 10  are also Raman spectra of Raman-active particles with MBA and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 11  are Raman spectra of Raman-active particles with trans-bis(pyridyl)ethylene (BPE) and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 12  are also Raman spectra of Raman-active particles with BPE and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 13  are Raman spectra of Raman-active particles with naphthalene thiol (NT) and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 14  are also Raman spectra of Raman-active particles with NT and SiO 2  coating in accordance with an embodiment of the invention; 
         FIG. 15  are Transmission Electron Microscopic (TEM) images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 50 nm in accordance with an embodiment of the invention; 
         FIG. 16  are TEM images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 30 nm in accordance with an embodiment of the invention; 
         FIG. 17  are TEM images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 15 nm in accordance with an embodiment of the invention; 
         FIG. 18  are TEM images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 50 nm in accordance with an embodiment of the invention; 
         FIG. 19  are TEM images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 30 nm in accordance with an embodiment of the invention; and 
         FIG. 20  are TEM images of Raman-active particles with MBA, SiO 2  coating, and core particles with an average size of 10 nm in accordance with an embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     Reference will now be made in detail to exemplary embodiments of the invention which are illustrated in the accompanying figures and examples. 
     With reference to  FIG. 2 , there is shown one embodiment of a Raman-active particle  110  that includes a core particle  112 , a coating  114 , and a Raman-active analyte  116 . It should be appreciated that one or more core particles  112 , coatings  114 , and analytes  116  may be included within the Raman-active particle  110 . The analyte  116  is at least partially within the coating  114  and the coating  114  substantially covers the core particle  112 . 
     In one embodiment, the core particle  112  has a metallic surface. The core particle  112  may include a metal selected from a group consisting of Au, Ag, Cu, Ni, Pd, Pt, Na, Al, and Cr, either individually or through any combination thereof. The core particle  112  may include any other inorganic or organic material provided the surface of the particle is metallic. In a particular embodiment, the core particle  112  comprises Au. 
     The shape of the core particle  112  may vary. For example, the core particle  112  may be in the shape of a sphere, fiber, plate, cube, tripod, pyramid, rod, tetrapod, or any non-spherical object. In one embodiment, the core particle  112  is substantially spherical. 
     The size of the core particle  112  also may vary and can depend on its composition and intended use. In one embodiment, the core particles  112  have an average diameter in a range from about 1 nm to about 500 nm. In another embodiment, the core particles  112  have an average diameter in a range from about 12 nm to about 100 nm. 
     The coating  114  includes a material whose function is to stabilize a Raman-active particle  110  against aggregation. The coating  114  stabilizes the Raman-active particle  110  in one way by inhibiting aggregation of Raman-active particle  10 . The coating  114  is sufficiently thick to stabilize the Raman-active particle  110 . In one embodiment, the coating  114  has a thickness in a range from about 1 nm to about 500 nm. In another embodiment, the coating  114  has a thickness in a range from about 5 nm to about 30 nm.  
     In one embodiment, the coating  114  comprises an elemental oxide. In a particular embodiment, the element in the elemental oxide includes silicon. The percentage of silicon may be varied and is dependent on several factors. Such factors may include the intended use of the Raman-active particle  110 , the composition of the core particle  112 , the degree to which the coating  114  is to be functionalized, the desired density of the coating  114  for a given application, the desired melting point for the coating  114 , the identity of any other materials which constitute the coating  114 , and the technique by which the Raman-active particle  110  is to be prepared. In one embodiment, the element in the elemental oxide of the coating  114  includes at least about 50-mole % silicon. In another embodiment, the element in the elemental oxide of the coating  114  includes at least about 30-mole % silicon. [Yet, in another embodiment, the element in the elemental oxide of the coating  114  comprises substantially silicon.] 
     In yet another embodiment, the coating  114  includes a composite coating. A composite coating comprises oxides of one or more elements selected from a group consisting of Si, B, Al, Ga, In, Sc, Y, La, Ti, Zr, Hf, V, Nb, Ta, Cr, Mn, Fe, Co, Ni, Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr, Ba, Zn, Cd, Ge, Sn, and Pb. Furthermore, the coating  114  may include multilayer coatings  114 . Each of the coating layers  114  in the multilayer coating  114  individually may include different coating compositions, such as 50-mole % silicon oxide in one coating layer and a composite coating in another coating layer. 
     The Raman-active particle  110  includes one or more Raman-active analytes  116 . The Raman-active analyte  116  is a molecule which exhibits Raman scattering when in the vicinity of a metallic surface of a core particle  112 . Examples of Raman-active analytes  116  include, but are not limited to, 4-mercaptopyridine, 2-mercaptopyridine (MP), trans-bis(pyridyl)ethylene (BPE), naphthalene thiol (NT), and mercaptobenzoic. The Raman-active analyte  116  may either individually include 4-mercaptopyridine, 2-mercaptopyridine, trans-bis(pyridyl)ethylene, naphthalene thiol, mercaptobenzoic acid or any combination thereof. 
     The Raman-active analyte  116  is at least partially within the coating  114 .  FIGS. 2-4  are schematic representations of the Raman-active analyte  116  at least partially within the coating  114 . The Raman-active analyte  116  can be at least partially within the coating  114  in various orientations, such as, but not limited to, dispersed within the coating  114  as in  FIG. 2 , within and around the coating  114  as in  FIG. 3 , or embedded within the coating  114  as in  FIG. 4 . Furthermore, a plurality of analytes  116  may be within the coating  114 . The plurality of analytes  116  may be within the coating  114  at a plurality of sites or at a single site. It should be appreciated that each of the analytes  116  may be within the coating  114  by a different mode, such as dispersed within the coating  114  as in  FIG. 2 , around the coating as in  FIG. 3 , or embedded within the coating  114  as in  FIG. 4 . 
     The Raman-active particle  110  may include one core particle  112  within a coating  114  as in  FIGS. 2-4  or multiple core particles  112  within a coating  114  as in  FIGS. 5-6 . The multiple core particles  112  are non-aggregated as in  FIG. 5  or closer together as in  FIG. 6 . There may be particular advantages associated with Raman-active particles  110  that have one core particle  112  within a coating  114  or multiple core particles  112  within a coating  114 . The selection as to how many core particles  112  should be contained within a coating  114  may depend largely on the particular application for which the Raman-active particles  110  are being used. Adjusting process conditions may be effective in obtaining Raman-active particles  110  with a single core particle  112  contained in the coating  114 . For example, the coating  114  may also stabilize a core particle  112  against aggregating with another core particle  112 . 
     The Raman-active particle  110  may vary in shape and size. In one embodiment, the Raman-active particles  110  are substantially spherical and have an average diameter in a range of up to about 1000 nm. 
     In one embodiment, the Raman-active particle  110  includes one or more linkers  118 , as in  FIG. 2 . The linker  118  binds to the core particle  112  and provides an interaction with the coating  114 . The linker  118  allows or facilitates the coating  114  to attach to the core particle  112 . The linker  118  may be a molecule comprising a functional group, which can bind to the metal surface of the core particle  112 , and a functional group onto which the coating  114  can deposit, such as alkoxysilanes. Examples of alkoxysilanes include trialkoxysilanes. Trialkoxysilane linkers  118  which may be used to deposit coatings  114  comprising silica include, but are not limited to, aminopropyl trimethoxysilane (APTMS), aminopropyl triethoxysilane, mercaptopropyl trimethoxysilane, mercaptopropyl triethoxysilane, hydroxypropyl trimethoxysilane, and hydroxypropyl triethoxysilane, either individually or through any combinations thereof. 
     When more than one analyte  116 , coating  114 , linker  118 , and core particle  112  are present, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of an analyte  116 , coating  114 , linker  118 , and core particle are permissible only if such combinations result in stable Raman-active particles  110 . Also, methods in the combinations of an analyte  116 , coating  114 , linker  118 , and core particle are permissible only if such combinations result in stable Raman-active particles  110 . 
     With reference to  FIGS. 7 and 8 , next will be described a method of making a Raman-active particle  110 .  FIG. 7  is a schematic representation of a method of making the Raman-active particle  110 .  FIG. 8  is a flow chart of method steps for making the Raman-active particle  110 . 
     The method includes, at step  805 , providing a colloidal solution comprising a core particle  112 . The core particle  112  may be an Au particle. The average size of the Au particles and amount of the colloidal solution may vary, such as for example, 50 mL of a 50 nm Au particles. The Au particle may be treated with ion exchange resin and filtered prior to beginning the coating reaction. 
     At step  815 , a coating  114  and or a coating precursor is provided to the colloidal solution comprising a core particle  112  to form a resulting solution. The resulting solution may either individually comprise a coating, a coating precursor, or a combination thereof. The coating  114  or coating precursor at least partially coats the core particle  112 . The coating precursor may be provided in the form of a sodium silicate solution or any other source of silica. 
     At step  825 , at least one Raman-active analyte  116  is provided to the resulting solution. The at least partial coating  114  of the core particle  112  is initiated before providing the Raman-active analyte  116 . However, the coating  114  does not have to be completed before providing the Raman-active analyte  116 . The providing of a coating  114  or coating precursor and providing of the Raman-active analyte  116  may occur simultaneously or overlap as the Raman-active analyte  116  may be provided concurrently with the completion of the coating  114 , but after the coating  114  is initiated. 
     As previously discussed, a linker  118  such as aminopropyl trimethoxysilane (APTMS) may be added to facilitate the deposition of the coating  114  onto the core particle  112 . The amino group of the aminopropyl trimethoxysilane binds to the surface of the core particle  112  while the alkoxysilane hydrolyzes, forming siloxy or hydroxy silyl groups. The hydrolyzed silane condenses with silicate in the silicate solution provided. In this way, the core particle  112  acts as a seed for growth of a silica coating. In one embodiment, a layer of silica coating  114  is deposited by adding a basic sodium silicate solution to an APTMS-modified colloidal gold core particle  112 . The high surface area of the APTMS-modified colloidal gold core particle  112  provides nucleation sites onto which the silicate coating  114  may deposit. This coating reaction using basic sodium silicate is referred to as the Water-glass reaction. The coating  114  may be made thicker using the Stober process in ethanol. 
     In another embodiment, a method of conducting an assay is provided. The method includes attaching a Raman-active particle  110  to one or more targeted moieties. Next, the Raman spectrum of the Raman-active particle  110  is measured. The Raman spectrum is then correlated to the presence of the targeted moiety. 
     The targeted moiety includes, but is not limited to biological species, small molecules, particles, viruses, peptides, DNA or RNA strands, and the like. 
     The following examples of Raman-active particles  101  with three varying average sizes of core particle  112 , three varying analytes, and two varying reactions are summarized in Table 1. The three varying average sizes of core particle  112  are 50 nm, 30 nm, and 15 nm of Au particles. The three varying analytes are MBA, BPE, and NT. The two reactions are Stober and Water Glass. 
     
       
         
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                 Size of Au core 
                   
                   
               
               
                   
                 Example 
                 particles (nm) 
                 Analyte 
                 Reaction 
               
               
                   
                   
               
             
             
               
                   
                 1A 
                 50 
                 MBA 
                 Stöber 
               
               
                   
                 1B 
                 30 
                 MBA 
                 Stöber 
               
               
                   
                 1C 
                 15 
                 MBA 
                 Stöber 
               
               
                   
                 2A 
                 50 
                 MBA 
                 Water Glass 
               
               
                   
                 2B 
                 30 
                 MBA 
                 Water Glass 
               
               
                   
                 2C 
                 15 
                 MBA 
                 Water Glass 
               
               
                   
                 3A 
                 50 
                 BPE 
                 Stöber 
               
               
                   
                 3B 
                 30 
                 BPE 
                 Stöber 
               
               
                   
                 3C 
                 15 
                 BPE 
                 Stöber 
               
               
                   
                 4A 
                 50 
                 BPE 
                 Water Glass 
               
               
                   
                 4B 
                 30 
                 BPE 
                 Water Glass 
               
               
                   
                 4C 
                 15 
                 BPE 
                 Water Glass 
               
               
                   
                 5A 
                 50 
                 NT 
                 Stöber 
               
               
                   
                 5B 
                 30 
                 NT 
                 Stöber 
               
               
                   
                 5C 
                 15 
                 NT 
                 Stöber 
               
               
                   
                 6A 
                 50 
                 NT 
                 Water Glass 
               
               
                   
                 6B 
                 30 
                 NT 
                 Water Glass 
               
               
                   
                 6C 
                 15 
                 NT 
                 Water Glass 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLES 1A-C 
     50 nm, 30 nm, and 15 nm Au Particles with MBA and SiO 2  Coating, under Stöber Reaction 
     A 50 mL colloidal solution comprising Au core particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 1A), 30 nm (Example 1B), and 15 nm (Example 1C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 400 μL of 0.62 mM 4-Mercaptobenzoic acid solution in ethanol was provided to the resulting solution. After stirring for 15 min, the solution was allowed to sit for 24 hours. The solution was poured into 180 mL EtOH with stirring, followed by 200 μL 30% ammonium hydroxide solution and 30 μL Si(OEt) 4 . The solution was stirred 15 min and let sit overnight. The solution was then placed into a flask and the solvent evaporated to a volume of approximately 30 mL, and then rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 9  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 1A-C with MBA analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
       FIG. 15-17  are TEM images of the embodiments of Raman-active particles  110  in Examples 1A-C respectively. The TEM images demonstrate that the Raman-active particles  110  are substantially non-aggregated and nanoscale sized. The Raman-active particles  110  also have a monomodal distribution typical of that observed in the preparation of gold colloids 
     EXAMPLES 2A-C 
     50 nm, 30 nm, and 15 nm Au particles with MBA and SiO 2  Coating, under Water Glass 
     A 50 mL colloidal solution comprising Au particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 2A), 30 nm (Example 2B), and 15 nm (Example 2C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 400 μL of 0.62 mM 4-Mercaptobenzoic acid solution in ethanol was provided to the resulting solution. After stirring for 15 min, the solution was allowed to sit for 7 days. The solution was rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 10  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 2A-C with MBA analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
       FIG. 18-20  are TEM images of the embodiments of Raman-active particles  110  in Example 2A-C respectively. The TEM images demonstrate that the Raman-active particles  110  are substantially non-aggregated and nanoscale sized. The Raman-active particles  110  also have a monomodal distribution typical of that observed in the preparation of gold colloids 
     EXAMPLES 3A-C 
     50 nm, 30 nm, and 15 nm Au Particles with BPE and SiO 2  Coating, under Stöber 
     A 50 mL colloidal solution comprising Au particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 3A), 30 nm (Example 3B), and 15 nm (Example 3C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 250 μL of 1.0 mM trans-bis(pyridyl) ethylene solution in ethanol was provided to the resulting solution. After stirring for 15 min, the resulting solution was allowed to sit for 24 hours. The solution was poured into 180 mL EtOH with stirring, followed by 200 μL 30% ammonium hydroxide solution and 30 μL Si(OEt) 4 . The solution was stirred 15 min and let sit overnight. The solution was then placed into a flask and the solvent evaporated to a volume of approximately 30 mL, and then rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 11  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 3A-C with BPE analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
     EXAMPLES 4A-C 
     50 nm, 30 nm, and 15 nm Au Particles with BPE and SiO 2  Coating, Under Water Glass 
     A 50 mL colloidal solution comprising Au particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 4A), 30 nm (Example 4B), and 15 nm (Example 4C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 250 μL of 1.0 mM trans-bis(pyridyl) ethylene solution in ethanol was provided to the resulting solution. After stirring for 15 min, the solution was allowed to sit for 7 days. The solution was rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 12  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 4A-C with BPE analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
     EXAMPLES 5A-C 
     50 nm, 30 nm, and 15 nm Au particles with NT and SiO 2  Coating, Under Stöber 
     A 50 mL colloidal solution comprising Au particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 5A), 30 nm (Example 5B), and 15 nm (Example 5C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 500 μL of 0.5 mM naphthalene thiol solution in ethanol was provided to the resulting solution. After stirring for 15 min, the resulting solution was allowed to sit for 24 hours. The solution was poured into 180 mL EtOH with stirring, followed by 200 μL 30% ammonium hydroxide solution and 30 μL Si(OEt) 4 . The solution was stirred 15 min and let sit overnight. The solution was then placed into a flask and the solvent evaporated to a volume of approximately 30 mL, and then rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 13  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 5A-C with NT analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
     EXAMPLE 6A-C 
     50 nm, 30 nm, and 15 nm Au Particles with NT and SiO 2  Coating, Under Water Glass 
     A 50 mL colloidal solution comprising Au particles was treated with 0.5 g of ion exchange resin for 30 min and filtered. The average sizes of the Au core particles were 50 nm (Example 6A), 30 nm (Example 6B), and 15 nm (Example 6C). The colloidal solution was placed in a beaker and 250 μL of 10 mM 3-aminopropyl trimethoxysilane solution was added dropwise followed by stirring for 15 minutes. Two mL of 0.54% sodium silicate solution was added slowly dropwise to the colloidal solution to form a resulting solution. 500 μL of 0.5 mM naphthalene thiol solution in ethanol was provided to the resulting solution. After stirring for 15 min, the solution was allowed to sit for 7 days. The solution was rinsed into a centrifuge tube and centrifuged for 1 hour. A pale pink supernatant liquid was decanted, leaving 3 mL total of a dark red colloid. 
       FIG. 14  are Raman spectra of the embodiments of the Raman-active particles  110  in Examples 6A-C with NT analyte and SiO 2  coating demonstrating the activeness of the Raman-active particles  110 . 
     While the invention has been described in detail in connection with only a limited number of aspects, it should be readily understood that the invention is not limited to such disclosed aspects. Rather, the invention can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the invention. Additionally, while various embodiments of the invention have been described, it is to be understood that aspects of the invention may include only some of the described embodiments. Accordingly, the invention is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.