Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS20040101889 A1
Publication typeApplication
Application numberUS 10/633,878
Publication dateMay 27, 2004
Filing dateAug 4, 2003
Priority dateJul 11, 2000
Also published asCA2415494A1, CA2415494C, DE60119170D1, DE60119170T2, EP1360318A2, EP1360318B1, US6602669, US20020034756, WO2002004681A2, WO2002004681A3
Publication number10633878, 633878, US 2004/0101889 A1, US 2004/101889 A1, US 20040101889 A1, US 20040101889A1, US 2004101889 A1, US 2004101889A1, US-A1-20040101889, US-A1-2004101889, US2004/0101889A1, US2004/101889A1, US20040101889 A1, US20040101889A1, US2004101889 A1, US2004101889A1
InventorsRobert Letsinger, Viswanadham Garimella
Original AssigneeNorthwestern University
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Method of detection by enhancement of silver staining
US 20040101889 A1
Abstract
The present invention relates to a method for amplifying a detection signal by enhancing or promoting the deposition of additional silver in assay detection systems where the formation of a silver spot serves as a reporter for the presence of a target molecule, including biological polymers (e.g., proteins and nucleic acids) and small molecules.
Images(5)
Previous page
Next page
Claims(30)
What we claim:
1. A method for amplifying a signal comprising:
(a) providing a substrate having deposited silver;
(b) contacting the substrate having deposited silver with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a substrate having a nanoparticles-silver sandwich;
(c) washing the substrate having said sandwich; and
(d) contacting the substrate having said sandwich with silver ions and a reducing agent to promote silver deposition onto the nanoparticles of said sandwich and produce silver-nanoparticles-silver sandwich.
(e) washing the substrate having the silver-nanoparticles-silver sandwich.
2. The method according to claim 1, wherein the nanoparticles comprise gold, silver, platinum or mixtures thereof.
3. The method according to claim 3, wherein the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticle-oligonucleotide conjugates or complexes.
4. The method according to claim 2, wherein the silver ion is derived from a silver salt comprising silver acetate, silver lactate, or silver nitrate.
5. The method according to claim 1 wherein the reducing agent comprises hydroquinone, n-propyl galate, p-phenylenediamine, or formaldehyde.
6. The method according to claim 1 wherein step (b) contacting is performed for a period ranging from about 5 to 30 minutes.
7. The method according to claim 6 wherein step (b) contacting is performed for about 10 minutes.
8. The method according to claim 1 wherein step (d) contacting is performed for a period ranging from about 5 to 10 minutes.
9. The method according to claim 8 wherein step (d) contacting is performed for about 5 minutes.
10. The method according to claim 1 wherein the deposited silver bound to the substrate is arranged in the form of an array.
11. The method according to claim 1, wherein step (c) washing is performed with water.
12. The method according to claim 1, wherein the substrate is glass.
13. A method for promoting silver deposition onto a surface comprising silver, said method comprising the steps of:
(a) providing a surface having silver bound thereto;
(b) contacting the surface with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a surface having a nanoparticles-silver sandwich;
(c) washing the surface having said nanoparticles-silver sandwich;
(d) contacting the surface having said nanoparticles-silver sandwich with a solution including silver ions under reducing conditions to promote silver deposition onto said nanoparticles of said nanoparticles-silver sandwich; and
(e) washing the surface having deposited silver.
14. The method according to claim 13 wherein said surface comprises cells or tissue.
15. The method according to claim 13, wherein the nanoparticles comprise gold, silver, platinum or combinations thereof.
16. The method according to claim 13, wherein the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticles having oligonucleotides bound thereto.
17. The method according to claim 16, wherein the gold nanoparticles having oligonucleotides bound thereto comprise gold nanoparticle-oligonucleotide conjugates or complexes.
18. The method according to claim 13, wherein the silver ion is derived from a silver salt comprising silver acetate, silver lactate, or silver nitrate.
19. The method according to claim 13 wherein the reducing agent comprising hydroquinone, n-propyl galate, p-phenylenediamine, or formaldehyde.
20. The method according to claim 13 wherein step (b) contacting is performed for a period ranging from about 5 to 30 minutes.
21. The method according to claim 20 wherein step (b) contacting is performed for a period of about 10 minutes.
22. The method according to claim 13 wherein step (d) contacting is performed for a period ranging from about 5 to 10 minutes.
23. The method according to claim 22 wherein step (d) contacting is performed for about 5 minutes.
24. The method according to claim 13 wherein the silver bound to the substrate are arranged in the form of an array.
25. A kit for signal amplification comprising:
(b) container including nanoparticles having oligonucleotides bound thereto;
(b) container including a silver salt; and
(c) container including a reducing agent.
26. The kit according to claim 25, wherein the nanoparticles comprise gold, silver, platinum or combinations thereof.
27. The kit according to claim 26, wherein the nanoparticles comprising gold nanoparticles having oligonucleotides bound thereto.
28. The kit according to claim 27, wherein the gold nanoparticles having oligonucleotides bound thereto comprise gold nanoparticle-oligonucleotide conjugates or complexes.
29. The kit according to claim 25, wherein the silver ion is derived from a silver salt comprising silver acetate, silver lactate, or silver nitrate.
30. The kit according to claim 25, wherein the reducing agent comprises hydroquinone, n-propyl galate, p-phenylenediamine, or formaldehyde.
Description
    CROSS-REFERENCE
  • [0001]
    This application claims the benefit of priority from U.S. Provisional application No. 60/217,782, filed Jul. 11, 2000.
  • FIELD OF THE INVENTION
  • [0002]
    The invention relates to several fields, especially to the detection of specific nucleic acids, proteins, carbohydrates, or organic compounds immobilized on a solid surface. More particularly it relates to detection systems in which the immobilized target is recognized by a metallic nanoparticle probe and for which the signal for detection has been amplified by reductive deposition of silver on the nanoparticle probe.
  • BACKGROUND OF THE INVENTION
  • [0003]
    (a) Gold Nanoparticle Probes:
  • [0004]
    The use of gold nanoparticle probes as reporter for detection of biological polymers was first described by W. P. Faulk and G. M. Taylor, who employed the nanoparticles as immunocytochemical probes for surface antigens [Immunochemitry, 8, 1081 (1971)]. Since then gold colloids have been widely used for detection of a variety of proteins using electron or light-microscopy to observe the particles [for reviews see Hacker, G. W. in Colloidal Gold; Principles, Methods, and Applications, Vol. 1, Academic Press, Inc. (1998) p 297, and Garzon, S., and Bendayan, M. in Immuno-Gold Electron Microscopy in Virus Diagnosis and Research, Ed. Hyatt, A. D. and Eaton, B. T., CRC Press, Ann Arbor, (1993) p 137]. Recently, applications of gold nanoparticle or cluster conjugates as probes for detection of oligonucleotides and nucleic acids have been suggested [Kidwell, D. A., and Conyers, S. M., U.S. Pat. No. 5,384,265 (1995); Hainfeld, J. F., et al. U.S. Pat. No. 5,521,289 (1996)] and described [Tomlinson, S., et al., Analytical Biochemistry, 171, 217 (1988); Mirkin et al., Nature, 15, 607 (1996); Storhoff, J. J. et al., J. Am. Chem. Soc., 120, 1959 (1998)].
  • [0005]
    (b) Silver Enhancement of Signal:
  • [0006]
    It has been found that the sensitivity for assays utilizing gold markers for proteins in tissues [Danscher, G. Histochemistry, 71, 1 (1981); Holgate, C. S. et al. J. Histochem. Cytochem. 31, 938 (1983)], for nucleic acids visualized in situ in immobilized biological systems [Gassell, G. J., et al., J. Cell Biology, 126, 863 (1994); Zehbe, I. et al., Am J. of Pathology, 150, 1553 (1997); Hacker, G. W., Eur. J. Histochem 42, 111 (1998) and for gold probes targeted to oligonucleotides captured on oligonucleotide arrays on a glass surface [T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 289, 1757 (2000)] can be significantly increased by silver staining. In this process, the gold particles captured on a surface are treated with a solution containing silver ions and a reducing agent (e.g., hydroquinone). The gold catalyzes reduction of the silver ions so that silver is deposited on the gold particle, and the early-deposited silver can itself catalyze further reduction of silver ion. As a consequence, the amount of metal that can be visualized is greatly increased. Unfortunately, however, the silver reduction catalyzed by the deposited silver ceases after a time, so the extent of amplification achievable is limited. When employed in enhancing visibility of gold nanoparticles on a glass plate, one observes darkening of the spot characteristic for the gold probes captured by a target sequence. Indeed, a good silver spot may be observed for cases where the amount of gold deposited initially is too small to be visible to the naked eye. Typically, the reaction time for the silver staining step is short, of the order of five minutes or less. Long exposure to the silver solution leads to non-selective deposition of silver metal and a high background. The silver ion solution and the reducing agent are mixed just prior to application to minimize the uncatalyzed reduction.
  • [0007]
    (c) Oligo- and Polynucleotide Arrays:
  • [0008]
    A recent major innovation in biology utilizes arrays of oligonucleotides or polynucleotides tethered to a solid surface. These oligomers serve as capture probes to bind complementary DNA or RNA target sequences. The captured sequences can in turn be recognized by fluorescent labels previously attached to them or by fluorescent or colorimetric probes that bind to a segment of the target. As stated by Eric Lander [Nature Genetics Supplement, 21, 3 (1999)]: “Arrays offer the first great hope . . . by providing a systematic way to survey DNA and RNA variation. They seem likely to become a standard tool of both molecular biology research and clinical diagnostics. These prospects have attracted great interest and investment from both the public and private sectors.”
  • [0009]
    Array technology is indeed now greatly accelerating developments in our understanding of genetic variation and gene expression. Nevertheless, current methodology suffers from several limitations, an important one being relatively low sensitivity in detecting fluorescently labeled targets on the chip arrays. Typically, targets in the range of picomolar concentrations or higher must be employed. Genetic analyses of natural targets in the attomolar or zeptomolar range therefore require target amplification by PCR. This procedure demands time and labor, and the target amplification can lead to errors in the sequence to be tested.
  • [0010]
    A need exits for a more sensitive, simpler, and cheaper detection method for polynucleotides arrayed on chips. Progress in detection technology has been made with the use of gold nanoparticle oligonucleotide conjugates as probes and signal amplification by silver ion reduction, which enables assays of polynucleotides of 50 fM concentration to be readily detected [for the methodology, see T. A. Taton, C. A. Mirkin, R. L. Letsinger,
  • [0011]
    Science, 289, 1757 (2000). We describe here a discovery that significantly lowers further the target concentration required for assays employing gold nanoparticles and other metallic nanoparticles.
  • SUMMARY OF THE INVENTION
  • [0012]
    The present invention relates to a method for amplifying signal by enhancing the deposition of silver in detecting systems where the formation of a silver spot serves as a reporter for the presence of a molecule, including biological polymers (e.g., proteins and nucleic acids) and small molecules. The detecting systems include detection of molecules in situ (e.g., on cells or in a tissue sample) and assays where the molecule to be detected (the target molecule) is bound to a substrate or is captured by another molecule bound to a substrate (the capture molecule). The invention has special utility in increasing the signal strength in diagnostic and screening applications involving detection of target molecules arrayed at discrete positions on a solid surface. It, therefore, provides a means for greatly enhancing the sensitivity of tests carried out on microarrays or microchips. The process is distinguished by the simplicity and economy of its execution and the large enhancement in signal and, thereby, sensitivity realized.
  • [0013]
    This invention is based on the discoveries that (1) gold nanoparticles coated with oligonucleotides bind to silver that had previously been deposited on gold nanoparticle-oligonucleotide conjugates immobilized by hybridization on a glass substrate or plate and (2) that the resulting (gold nanoparticle-oligonucleotide-silver-(gold-oligonucleotide) structures function as catalyst for the further deposition of silver by reduction of silver ions. The first discovery is surprising since one might expect that the surface bound oligonucleotides, which shield the nanoparticles from non-specific binding to the glass surface and the oligonucleotides immobilized on the glass surface, would also shield the nanoparticles against interaction with the silver surface. Indeed, other work has shown that oligonucleotides protect gold nanoparticle oligonucleotide conjugates from fusing to form gold-gold bonds between individual nanoparticles even when the mixtures are dried. The second discovery is significant since it provides a means for substantially increasing the metallic mass at the site of the originally immobilized nanoparticles. In conjunction with development of buffer conditions that enable oligonucleotide nanoparticle conjugates that are unbound by hybridization or interaction with silver to be washed cleanly from the glass surface these findings opened opportunities for assaying polynucleotide targets at extremely low target concentrations.
  • [0014]
    Accordingly, one objective of the invention is to provide a method for amplifying a detection signal comprising:
  • [0015]
    (a) providing a substrate having deposited silver;
  • [0016]
    (b) contacting the substrate having deposited silver with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a substrate having a nanoparticles-silver sandwich;
  • [0017]
    (c) washing the substrate having said sandwich; and
  • [0018]
    (d) contacting the substrate having said sandwich with silver ions and a reducing agent to promote silver deposition onto the nanoparticles of said sandwich.
  • [0019]
    The nanoparticles having oligonucleotides bound thereto comprise gold, silver, platinum or mixtures thereof. These nanoparticles may be in the form of gold nanoparticle-oligonucleotide conjugates or complexes.
  • [0020]
    Another object of the invention is to provide a method for promoting silver deposition onto a surface comprising silver, said method comprising the steps of:
  • [0021]
    (a) providing a surface having silver bound thereto;
  • [0022]
    (b) contacting the surface with a solution comprising nanoparticles having oligonucleotides bound thereto so as to produce a surface having a nanoparticles-silver sandwich;
  • [0023]
    (c) washing the surface having said nanoparticles-silver sandwich;
  • [0024]
    (d) contacting the surface having said nanoparticles-silver sandwich with a solution including silver ions under reducing conditions to promote silver deposition onto said nanoparticles of said nanoparticles-silver sandwich; and
  • [0025]
    (e) washing the surface having deposited silver.
  • [0026]
    According to this method, the surface may include cells or tissue for in situ detection of target molecules. Preferrably the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticles having oligonucleotides bound thereto in conjugate or complex form. In practicing this invention, gold nanoparticle oligonucleotide conjugates are preferred.
  • [0027]
    A further object of the invention is to provide a kit for detection signal amplification comprising:
  • [0028]
    (a) container including nanoparticles having oligonucleotides bound thereto;
  • [0029]
    (b) container including a silver salt; and
  • [0030]
    (c) container including a reducing agent.
  • [0031]
    The kit may include instructions for use in amplifying silver stain detection signals. Preferrably the nanoparticles having oligonucleotides bound thereto comprise gold nanoparticles having oligonucleotides bound thereto in conjugate or complex form. In practicing this invention, gold nanoparticle oligonucleotide conjugates are preferred.
  • DESCRIPTION OF THE FIGURES
  • [0032]
    [0032]FIG. 1 is a schematic representation of amplification of a detection signal for a gold nanoparticle immobilized on a solid surface. Step (a) is conventional silver staining. In step (c), gold nanoparticles bearing oligonucleotides are bound to the silver surface. Step (e) is conventional silver staining of the gold nanoparticles that were bound to the previous silver surface.
  • [0033]
    [0033]FIG. 2 illustrates two plates with or without application of the amplification method of the invention. Plate (a) was subjected to a second round of conventional silver staining in an attempt to further enhance the spot. Slight but very little signal enhancement took place. Plate (b) was subjected to the signal amplification method of the invention.
  • [0034]
    [0034]FIG. 3 illustrates two plates with or without application of the amplification method of the invention. Plate (a) indicates the signal obtained for a sample using conventional silver staining. Plate (b) shows the enhancement achievable by the application of the inventive signal amplification method.
  • [0035]
    [0035]FIG. 4 illustrates two plates with or without application of the amplification method of the invention. Plate (a) shows the results of conventional silver staining for a test in which a 25 fM solution of the target oligonucleotide was used. Plate (b) shows the results for the same test in which the initial silver staining was followed with the signal amplification method of the invention.
  • [0036]
    [0036]FIG. 5 illustrates a plate following application of the inventive signal amplification method of the invention. Spot obtained after two cycles of the nanoparticle-oligonucleotide (conjugate I)/silver staining methodology applied when gold nanoparticle probes and an oligonucleotide target (63-mer) were immobilized on a glass plate. The concentration of the target oligonucleotide in this case was only 1 fM.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0037]
    The present invention relates to a method for signal amplification for detecting target substances. In particular, the invention provides for amplification of signal and enhancement of assay sensitivity for detection of minute quantities of target molecules, e.g., nucleic acids, that are based on silver stain detection. In practicing this invention, the inventive method is used after the application of conventional silver staining where it leads to the formation of silver-gold(oligonucleotide)-silver assemblies, termed here silver-gold′-silver sandwich assemblies.
  • [0038]
    The prior steps leading up to and including conventional silver staining can, for example, involve: capture of a target oligonucleotide by an oligonucleotide capture probe bound to a glass surface, washing, addition of nanoparticle oligonucleotide conjugates complementary to an unhybridized segment of the bound target oligonucleotide, washing, addition of a silver staining solution (Ag+ plus a reducing agent, available through Sigma), washing, drying, and viewing, either with the naked eye or with aid of pictures obtained using a simple flat-bed scanner. This procedure was described by Taton et al., Science, 289, 1757 (2000), who showed that although the gold spot obtained was too weak to observe directly for target concentrations of 100 nM in oligonucleotide, conventional silver deposition affords a strong dark spot. The limit of detection for this system using conventional silver staining is 50 fM concentrations, for which an extremely faint spot is observed. Re-exposure to the silver solution did not noticeably further enhance the intensity of the spot.
  • [0039]
    For amplification of the silver signal by the inventive silver-gold′-silver sandwich method, the glass plate containing silver stain from a prior application of conventional staining is further exposed to an aqueous amplification solution of nanoparticles having oligonucleotides bound thereto, preferably gold nanoparticle-oligonucleotide conjugates or a gold-nanoparticle oligonucleotide complexes. The oligonucleotide sequence does not need to be related to the target oligonucleotide sequence or the sequences on the initial probes. For gold nanoparticle-oligonucleotide conjugates, the amplification solution generally contains between about 1 nM and about 5 nM nanoparticle oligonucleotide conjugates. The amplification solution may include salts, such as buffer salts, and detergents such as phosphate, citrate, HEPES, and NES and is preferably has a pH of about 7. In practicing the invention, an aqueous amplification solution including aqueous 0.1M NaCl and 10 mM phosphate (pH 7.0) was found to be particularly useful. For gold nanoparticle oligonucleotide complexes, the amplification solution is the same. Any suitable mode of exposing the silver stain to the amplification solution may be used. Representative examples include spraying, dipping, and the like.
  • [0040]
    After about 5 to 30 minutes, preferably about 10 minutes, the unbound nanoparticles conjugates or complexes are removed by washing the plate with a suitable aqueous solution, preferably water. Optionally, the plate is dried by any suitable method such as air drying. The plate is then re-exposed to a silver staining solution for a suitable time period, generally for about 5 to about 10 minutes, preferably about 5 minutes. It will be understood that any suitable silver staining method may be used. Suitable, but non-limiting, examples of useful silver staining methods and compositions are described, for instance, in M. A. Hayat, Ed., “Immunogold-Silver Staining,” CRC Press (1995). Generally, silver staining solutions include silver ion in the form of a silver salt such as silver acetate (preferred), silver lactate, and silver nitrate. These solutions also include a reducing agent that is admixed into the solution just prior to use. Suitable, but non-limiting, examples of reducing agents include hydroquinone, n-propyl galate, p-phenylenediamine, and formaldehyde. If desired, other agents such as gum Arabic may be used to mediate the silver stain process. Any suitable mode of contacting the substrate with the silver stain solution may be used. Representative examples include spraying, dipping, and the like.
  • [0041]
    Following washing with water (preferred) or other suitable solution (e.g., aqueous solution containing 0.1M NaCl and 10 mM phosphate (pH 7.0) to remove unreacted silver stain solution, the re-exposed plate is observed visually or copied by a flat-bed scanner. This treatment leads to a great increase in the darkness of the spot. The process may be repeated as often as desired to further enhance the amount of deposited silver and the darkness of the spot. With this inventive amplification method, one can readily observe a dark silver spot for an assay using 25 μM target concentration. With two cycles of the new nanoparticle-silver sandwich procedure, 1 fM target solutions can be recognized, and with three cycles, 0.1 fM solutions give positive though weak spots. These experiments were carried out using 1 microliter of the target solution in each case. For the assay with 0.1 fM solution this corresponds to 60 target molecules in the applied sample.
  • [0042]
    Any substrate can be used which allows observation of a silver stain as the detectable change. Suitable substrates include transparent solid surfaces (e.g., glass, quartz, plastics and other polymers. The substrate can be any shape or thickness, but generally will be flat and thin. Preferred are transparent substrates such as glass (e.g., glass slides) or plastics (e.g., wells of microtiter plates).
  • [0043]
    The silver stain signal amplification method of the invention depends on the use of nanoparticles-oligonucleotide conjugates or complexes that satisfy certain characteristics. First, the nanoparticles do not stick to the surface of the chip being tested. Ordinary nanoparticles prepared by the citrate reduction method of Frens (Frens, G., Nat. Phys. Sci., 241, 20-22 (1973) are not satisfactory since they bind indiscriminately to the oligonucleotide-derivatized glass plate used as the substrate for these assays. Subsequent silver enhancement then gives false positives as dark areas. Second, the nanoparticles bind to a deposited silver surface such that on subsequent washing, the attached nanoparticles remain bound to the silver area while nanoparticles suspended in solution are cleanly removed. Third, the nanoparticles function as agents to reduce silver ions under silver staining conditions. In practicing this invention, useful nanoparticles are nanoparticles coated with oligonucleotides linked through sulfur to the surface (nanoparticle oligonucleotide conjugates) such as the ones described in J. J. Storhoff et al., J. Am. Chem. Soc., 120, 1958 (1999) (for a specific example, see conjugate I in Example 1 below) or with natural type oligonucleotides adsorbed to the surface (nanoparticle oligonucleotide complexes) such as conjugate III described in Example 1. Both types of nanoparticles work well in low or moderate salt solution (e.g. up to 0.1 M), but the conjugates containing the sulfur anchor are particularly preferred for tests conducted at high salt concentration, at which the complexes formed by simple adsorption of oligonucleotides are unstable and aggregate. It will be understood by the ordinary skilled artisan that any nanoparticle preparation that meets the criteria listed above are useful as intermediary agents in forming the sandwich assemblies, and the methodology can be applied for the amplification of the silver signal for any target visualized by an initial silver deposition. While gold nanoparticles are particularly preferred, any nanoparticle that catalyze the reduction of silver can be used including silver and platinum nanoparticles.
  • [0044]
    The preparation of nanoparticles suitable for use in the practice of the invention, the attachment of oligonucleotides to them, the flatbed scanner technique, and various assays formats for the detection of nucleic acids using conventional silver staining are described in co-pending application Ser. No. 09/760,500, filed Jan. 12, 2001; Ser. No. 09/603,830, filed Jun. 26, 2000; Ser. No. 09/344,667, filed Jun. 25, 1999; Ser. No. 09/240,755, filed Jan. 29, 1999; 60/031,809, filed Jul. 29, 1996; 60/176,409; and 60/200,161, filed Apr. 26, 2000; and international application Nos. PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26, 2000; and PCT/US01/01190, filed Jan. 12, 2001, entitled “Nanoparticles Having Oligonucleotides Attached Thereto And Uses Therefor,” the entire contents of which are incorporated herein by reference.
  • EXAMPLES Example 1 Preparation of Oligonucleotide Modified Gold Nanoparticles.
  • [0045]
    Oligonucleotides and 5′-mercaptoalkyl-oligonucleotides were prepared using phosphoramidite chemistry as described by Storhoff et al. [J. Am. Chem. Soc. 120, 1959-1964 (1998)]. Gold nanoparticles (˜13 nm diameter) were prepared as described by Grabar, K. C., et al. [J. Analyt. Chem., 67, 735-743 (1995); Frens, G., Nat. Phys. Sci., 241, 20-22 (1973)].
  • [0046]
    For preparation of the nanoparticle conjugate (I) used in the sandwich silver-gold′-silver amplification scheme, 5′-mercaptoalkyl-oligonucleotide II was prepared and joined to gold nanoparticles by the general linking procedure described by Storhoff et al., [J. Am. Chem. Soc. 120, 1959-1964 (1998)]. The nanoparticle-oligonucleotide complex (m) was prepared by mixing 1 μL of an aqueous solution containing 0.26 A260 Units of oligonucleotide IV with 100 μL of citrate stabilized gold colloid (prepared as described by Grabar et al. [J. Analyt. Chem., 67, 735-743 (1995)) and allowing the solution to stand overnight.
  • [0047]
    I. Conjugate formed from gold nanoparticles and II.
  • [0048]
    II. 5′-HS(CH2)6OP(O)(O)O-(A)20TGGGTAGCAGACCTC (SEQ ID NO.: 1)
  • [0049]
    III. Complex formed from gold nanoparticles and IV
  • [0050]
    IV. 5′-GCTCTAGAATGAACGGTGGAAGGCGGCAGG (SEQ ID NO.:2)
  • Example 2 Sandwich Amplification of Silver Signal
  • [0051]
    In this Example, four separate experiments were conducted using the nanoparticle oligonucleotide conjugate (I) or complex (II) prepared as described in Example 1, and glass slides containing silver spots from oligonucleotide assays carried out using the silver staining method described by T. A. Taton et al, Science, 289, 1757-60 (2000). The glass plate bearing the silver spots was exposed to a solution of gold nanoparticle conjugate I (FIGS. 2, 4, and 5) or gold nanoparticle complex III (FIG. 3) for 10 minutes, washed with 1 M NaNO3 in nanopure water, and exposed for five minutes at room temperature to a 1:1 mixture of freshly mixed sample of the two commercial Silver Enhancer solutions (Catalog Nos. 55020 and 55145, Sigma Corporation, St. Louis, Mo.) for 5 minutes, following the Sigma protocol for the silver staining step, including final washes with nanopure water, sodium thiosulfate solution, and nanopure water. The plate was dried and observed, both visually and by copying for records using a Hewlett Packard Scanner [Model no. 5200C]. The direct visual observations of spots corresponded to the prints obtained using the scanner. The figures are enlarged.
  • [0052]
    [0052]FIG. 2 illustrates the results for the first experiment using nanoparticle oligonucleotide conjugate I. In this experiment, two plates, each containing very faint silver spots from previous hybridization and silver staining of a dilute target solution, were used. Plate (a) was subjected to a second round of conventional silver staining in an attempt to further enhance the spot. Slight but very little signal enhancement took place. Plate (b) was subjected to conditions for the gold sandwich amplification. This entailed exposing the plate containing the weak silver spots to the solution of nanoparticle conjugate I for 10 minutes, followed by washing with water and conventional silver staining for 4 minutes. The darkness of the spots in plate (b) relative to those in plate (a) clearly demonstrate the power of the new metal sandwich or signal enhancement method.
  • [0053]
    [0053]FIG. 3 illustrates the results for an experiment using nanoparticle oligonucleotide complex III. Plate (a) indicates the signal obtained for a sample using conventional silver staining. Plate (b) shows the enhancement achievable by the gold sandwich technology. Note that two sample spots are shown in each case. For plate (b), a plate containing a weak silver spot corresponding to that in plate (a) was exposed to a solution of nanoparticle oligonucleotide complex III, and following washing, was subjected to conventional silver staining. In this case, three parts of colloid III, prepared as described above, was mixed with seven parts of 10 mM phosphate buffer at pH 7. The plate was then exposed to this colloid solution for 10 minutes, washed with nanopure water, and exposed to the silver staining mixture for 4 minutes as before.
  • [0054]
    [0054]FIG. 4 illustrates the results for an experiment using gold nanoparticle oligonucleotide I to enhance the weak silver signal resulting from an assay of a 63 nucleotide target oligonucleotide
  • [0055]
    5′ GTA GGC GAA CCC TGC CCA GGT CGA CAC ATA GG T GAG GTC TGC TAC CCA CAG CCG GTT AGG TGC 3′ (SEQ ID NO.: 3)
  • [0056]
    at 25 fM concentration. Note that our standard assay using just silver amplification gives a very weak spot (plate (a)). Plate (a) shows the results of conventional silver staining for a test in which a 25 fM solution of the target oligonucleotide was used. Plate (b) shows the results for the same test in which the initial silver staining was following by 10 minute exposure to the colloidal solution of nanoparticle conjugate I, followed by silver staining. In this case, the gold solution had been diluted six-fold with 0.1 M NaCl-10 mM phosphate buffer. Note the strong signal for a target, captured at 25 fM concentration, after the metal sandwich enhancement procedure.
  • [0057]
    [0057]FIG. 5 illustrates the results for an experiment using nanoparticle oligonucleotide conjugate I to enhance the silver signal obtained from an assay carried out using a 63-mer target [see SEQ ID NO.: 3] at a concentration of 1 fM. In this case, our standard procedure for silver staining failed to show any spot. Double enhancement using the gold-silver treatment, however, showed a strong signal, as shown. This experiment was carried out by treating the plate that had been exposed to silver amplification by the stand procedure successively with: (1) gold nanoparticle conjugate I (1.5 nM in nanoparticles in 0.1 M NaCl and 10 mM phosphate buffer at pH 7.0) for 10 minutes; (2) wash with 1 M NaNO3; (3) treat with a mixture of Silver Enhancer solutions (catalog nos. 5020 and S 5145, Sigma Corporation, by the Sigma protocol; (4) wash with 1 M sodium nitrate; (5) repeat steps 1-4; (6) wash with sodium thiosulfate; and (7) wash with water.
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US4193983 *May 16, 1978Mar 18, 1980Syva CompanyLabeled liposome particle compositions and immunoassays therewith
US4256834 *Apr 9, 1979Mar 17, 1981Syva CompanyFluorescent scavenger particle immunoassay
US4261968 *May 10, 1979Apr 14, 1981Syva CompanyFluorescence quenching with immunological pairs in immunoassays
US4313734 *Jul 13, 1979Feb 2, 1982Akzona IncorporatedMetal sol particle immunoassay
US4318707 *Jun 3, 1980Mar 9, 1982Syva CompanyMacromolecular fluorescent quencher particle in specific receptor assays
US4650770 *Dec 7, 1983Mar 17, 1987Syntex (U.S.A.) Inc.Energy absorbing particle quenching in light emitting competitive protein binding assays
US4713348 *Oct 22, 1985Dec 15, 1987Syntex (U.S.A.) Inc.Fluorescent multiparameter particle analysis
US4853335 *Sep 28, 1987Aug 1, 1989Olsen Duane AColloidal gold particle concentration immunoassay
US4868104 *Sep 6, 1985Sep 19, 1989Syntex (U.S.A.) Inc.Homogeneous assay for specific polynucleotides
US4996143 *Apr 13, 1990Feb 26, 1991Syngene, Inc.Fluorescent stokes shift probes for polynucleotide hybridization
US5225064 *Mar 6, 1992Jul 6, 1993Enzyme Technology Research Group, Inc.Peroxidase colloidal gold oxidase biosensors for mediatorless glucose determination
US5284748 *Apr 10, 1992Feb 8, 1994Immunotronics, Inc.Method for electrical detection of a binding reaction
US5288609 *Oct 30, 1992Feb 22, 1994Enzo Diagnostics, Inc.Capture sandwich hybridization method and composition
US5294369 *Dec 5, 1990Mar 15, 1994Akzo N.V.Ligand gold bonding
US5360895 *Dec 9, 1992Nov 1, 1994Associated Universities, Inc.Derivatized gold clusters and antibody-gold cluster conjugates
US5384073 *Jan 21, 1994Jan 24, 1995Akzo N.V.Ligand gold bonding
US5384265 *Mar 26, 1993Jan 24, 1995Geo-Centers, Inc.Biomolecules bound to catalytic inorganic particles, immunoassays using the same
US5460831 *Nov 4, 1993Oct 24, 1995The Regents Of The University Of CaliforniaTargeted transfection nanoparticles
US5472881 *Mar 21, 1994Dec 5, 1995University Of Utah Research FoundationThiol labeling of DNA for attachment to gold surfaces
US5508164 *Oct 29, 1993Apr 16, 1996Dekalb Genetics CorporationIsolation of biological materials using magnetic particles
US5514602 *Feb 25, 1993May 7, 1996Ortho Diagnostic Systems, Inc.Method of producing a metal sol reagent containing colloidal metal particles
US5521289 *Jul 29, 1994May 28, 1996Nanoprobes, Inc.Small organometallic probes
US5543158 *Jul 23, 1993Aug 6, 1996Massachusetts Institute Of TechnologyBiodegradable injectable nanoparticles
US5571726 *May 19, 1995Nov 5, 1996Ortho Diagnostic Systems, Inc.Kit containing glutaraldehyde coated colloidal metal particles of a preselected size
US5599668 *Sep 22, 1994Feb 4, 1997Abbott LaboratoriesLight scattering optical waveguide method for detecting specific binding events
US5609907 *Feb 9, 1995Mar 11, 1997The Penn State Research FoundationSelf-assembled metal colloid monolayers
US5637508 *Jan 23, 1995Jun 10, 1997Geo-Centers, Inc.Biomolecules bound to polymer or copolymer coated catalytic inorganic particles, immunoassays using the same and kits containing the same
US5665582 *Apr 18, 1994Sep 9, 1997Dekalb Genetics Corp.Isolation of biological materials
US5681943 *May 8, 1995Oct 28, 1997Northwestern UniversityMethod for covalently linking adjacent oligonucleotides
US5751018 *Apr 29, 1994May 12, 1998The Regents Of The University Of CaliforniaSemiconductor nanocrystals covalently bound to solid inorganic surfaces using self-assembled monolayers
US5830986 *Oct 28, 1996Nov 3, 1998Massachusetts Institute Of TechnologyMethods for the synthesis of functionalizable poly(ethylene oxide) star macromolecules
US5900481 *Nov 6, 1996May 4, 1999Sequenom, Inc.Bead linkers for immobilizing nucleic acids to solid supports
US5922537 *Nov 8, 1996Jul 13, 1999N.o slashed.AB Immunoassay, Inc.Nanoparticles biosensor
US5939021 *Jan 23, 1997Aug 17, 1999Hansen; W. PeterHomogeneous binding assay
US5972615 *Jan 21, 1998Oct 26, 1999Urocor, Inc.Biomarkers and targets for diagnosis, prognosis and management of prostate disease
US5990479 *Nov 25, 1997Nov 23, 1999Regents Of The University Of CaliforniaOrgano Luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes
US6025202 *Dec 16, 1998Feb 15, 2000The Penn State Research FoundationSelf-assembled metal colloid monolayers and detection methods therewith
US6149868 *Oct 28, 1998Nov 21, 2000The Penn State Research FoundationSurface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches
US6203989 *Mar 25, 1999Mar 20, 2001Affymetrix, Inc.Methods and compositions for amplifying detectable signals in specific binding assays
US6214560 *Apr 18, 1997Apr 10, 2001Genicon Sciences CorporationAnalyte assay using particulate labels
US6251303 *Sep 18, 1998Jun 26, 2001Massachusetts Institute Of TechnologyWater-soluble fluorescent nanocrystals
US6264825 *Jun 23, 1999Jul 24, 2001Clinical Micro Sensors, Inc.Binding acceleration techniques for the detection of analytes
US6277489 *Dec 4, 1998Aug 21, 2001The Regents Of The University Of CaliforniaSupport for high performance affinity chromatography and other uses
US6306610 *Sep 17, 1999Oct 23, 2001Massachusetts Institute Of TechnologyBiological applications of quantum dots
US6361944 *Jun 25, 1999Mar 26, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6365418 *May 18, 2000Apr 2, 2002Zyomyx, IncorporatedArrays of protein-capture agents and methods of use thereof
US6417340 *Oct 20, 2000Jul 9, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6495324 *Oct 20, 2000Dec 17, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6506564 *Jun 26, 2000Jan 14, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6582921 *Sep 20, 2001Jun 24, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses thereof
US6602669 *Jul 11, 2001Aug 5, 2003Northwestern UniversityMethod of detection by enhancement of silver staining
US6610491 *Sep 28, 2001Aug 26, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6645721 *Sep 20, 2001Nov 11, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6673548 *Sep 28, 2001Jan 6, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6677122 *Oct 11, 2001Jan 13, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6682895 *Oct 12, 2001Jan 27, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6709825 *Oct 10, 2001Mar 23, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6720147 *Oct 12, 2001Apr 13, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6720411 *Oct 10, 2001Apr 13, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6726847 *Nov 30, 2001Apr 27, 2004Northwestern UniversitySilver stain removal by chemical etching and sonication
US6730269 *Oct 12, 2001May 4, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6740491 *Sep 28, 2001May 25, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6750016 *Mar 28, 2001Jun 15, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6759199 *Sep 20, 2001Jul 6, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6767702 *Jan 12, 2001Jul 27, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6773884 *Aug 7, 2001Aug 10, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6777186 *Oct 15, 2001Aug 17, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020137070 *Oct 10, 2001Sep 26, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020155461 *Oct 12, 2001Oct 24, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020160381 *Oct 11, 2001Oct 31, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020177143 *Dec 28, 2001Nov 28, 2002Mirkin Chad A.Non-alloying core shell nanoparticles
US20020192687 *Mar 27, 2002Dec 19, 2002Mirkin Chad A.Bio-barcodes based on oligonucleotide-modified nanoparticles
US20030054358 *Oct 11, 2001Mar 20, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030068638 *Aug 2, 2002Apr 10, 2003William CorkNanoparticle imaging system and method
US20030087242 *Dec 7, 2001May 8, 2003Mirkin Chad A.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030113740 *Apr 18, 2002Jun 19, 2003Mirkin Chad A.Oligonucleotide-modified ROMP polymers and co-polymers
US20030124528 *Oct 12, 2001Jul 3, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030129608 *May 22, 2002Jul 10, 2003Mirkin Chad ANon-alloying core shell nanoparticles
US20030143538 *Oct 11, 2001Jul 31, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030143598 *Nov 8, 2002Jul 31, 2003Viswanadham GarimellaBioconjugate-nanoparticle probes
US20030148282 *Oct 12, 2001Aug 7, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030207296 *Oct 8, 2002Nov 6, 2003So-Jung ParkNanoparticles having oligonucleotides attached thereto and uses therefor
US20030211488 *Jun 14, 2002Nov 13, 2003Northwestern UniversityNanoparticle probs with Raman spectrocopic fingerprints for analyte detection
US20040038255 *Mar 26, 2003Feb 26, 2004Northwestern UniversityNon-alloying core shell nanoparticles
US20040053222 *Jul 2, 2003Mar 18, 2004Nanosphere, Inc.Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes
US20040072231 *Aug 13, 2003Apr 15, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20040086897 *May 7, 2003May 6, 2004Mirkin Chad A.Nanoparticle probes with Raman Spectroscopic fingerprints for analyte detection
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US6962786Apr 9, 2003Nov 8, 2005Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6969761Oct 12, 2001Nov 29, 2005Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US6974669Mar 27, 2002Dec 13, 2005Nanosphere, Inc.Bio-barcodes based on oligonucleotide-modified nanoparticles
US6986989Oct 12, 2001Jan 17, 2006Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7098320Oct 11, 2001Aug 29, 2006Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7169556Oct 8, 2002Jan 30, 2007Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7186814Nov 8, 2002Mar 6, 2007Nanosphere, Inc.Bioconjugate-nanoparticle probes
US7208587Aug 13, 2003Apr 24, 2007Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7250499Nov 18, 2003Jul 31, 2007Nanosphere Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7253277Jul 2, 2003Aug 7, 2007Nanosphere, Inc.Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes
US7259252Oct 10, 2001Aug 21, 2007Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US7985539May 7, 2003Jul 26, 2011Northwestern UniversityNanoparticle probes with raman spectroscopic fingerprints for analyte detection
US8323888Feb 2, 2007Dec 4, 2012Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US8609433Dec 2, 2010Dec 17, 2013Rapid Pathogen Screening, Inc.Multiplanar lateral flow assay with sample compressor
US8815609Mar 7, 2013Aug 26, 2014Rapid Pathogen Screening, Inc.Multiplanar lateral flow assay with diverting zone
US8962260Mar 8, 2013Feb 24, 2015Rapid Pathogen Screening, Inc.Method and device for combined detection of viral and bacterial infections
US9068981Sep 19, 2012Jun 30, 2015Rapid Pathogen Screening, Inc.Lateral flow assays with time delayed components
US20020137071 *Oct 10, 2001Sep 26, 2002Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020192687 *Mar 27, 2002Dec 19, 2002Mirkin Chad A.Bio-barcodes based on oligonucleotide-modified nanoparticles
US20030054358 *Oct 11, 2001Mar 20, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030068622 *Oct 12, 2001Apr 10, 2003Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20030113740 *Apr 18, 2002Jun 19, 2003Mirkin Chad A.Oligonucleotide-modified ROMP polymers and co-polymers
US20030129608 *May 22, 2002Jul 10, 2003Mirkin Chad ANon-alloying core shell nanoparticles
US20030207296 *Oct 8, 2002Nov 6, 2003So-Jung ParkNanoparticles having oligonucleotides attached thereto and uses therefor
US20030211488 *Jun 14, 2002Nov 13, 2003Northwestern UniversityNanoparticle probs with Raman spectrocopic fingerprints for analyte detection
US20040038255 *Mar 26, 2003Feb 26, 2004Northwestern UniversityNon-alloying core shell nanoparticles
US20040053222 *Jul 2, 2003Mar 18, 2004Nanosphere, Inc.Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes
US20040110220 *Nov 18, 2003Jun 10, 2004Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20050037397 *Jun 25, 2004Feb 17, 2005Nanosphere, Inc.Bio-barcode based detection of target analytes
US20050130174 *Feb 27, 2004Jun 16, 2005Nanosphere, Inc.Label-free gene expression profiling with universal nanoparticle probes in microarray assay format
US20050250094 *Nov 22, 2004Nov 10, 2005Nanosphere, Inc.Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes
US20060014172 *May 3, 2005Jan 19, 2006Nanosphere, Inc.Aptamer-nanoparticle conjugates and method of use for target analyte detection
US20060040286 *May 12, 2005Feb 23, 2006Nanosphere, Inc.Bio-barcode based detection of target analytes
US20060057613 *Jul 26, 2005Mar 16, 2006Nanosphere, Inc.Method for distinguishing methicillin resistant S. aureus from methicillin sensitive S. aureus in a mixed culture
US20060068378 *Feb 4, 2005Mar 30, 2006Nanosphere, Inc.Nanoparticles having oligonucleotides attached thereto and uses therefor
US20070154903 *Jun 23, 2006Jul 5, 2007Nanosphere, Inc.Selective isolation and concentration of nucleic acids from complex samples
US20070190551 *Dec 8, 2006Aug 16, 2007Nanosphere, Inc.Non-alloying core shell nanoparticles
US20090111094 *Aug 18, 2006Apr 30, 2009Nanosphere, Inc.Methods for preparing hybrid substrates comprising DNA and antibodies and uses thereof
US20110136258 *Dec 2, 2010Jun 9, 2011Rapid Pathogen Screening, Inc.Multiplanar Lateral Flow Assay with Sample Compressor
Classifications
U.S. Classification435/6.12
International ClassificationG01N33/53, G01N37/00, G01N33/58, G01N33/543, C12N15/09, C12Q1/68, G01N27/447
Cooperative ClassificationY10S977/924, B82Y15/00, G01N27/44721, G01N27/44726, C12Q2563/137
European ClassificationB82Y15/00, G01N27/447B3A2, G01N27/447B3A
Legal Events
DateCodeEventDescription
May 13, 2015ASAssignment
Owner name: NORTHWESTERN UNIVERSITY, ILLINOIS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LETSINGER, ROBERT L.;GARIMELLA, VISWANADHAM;NANOSPHERE, INC.;SIGNING DATES FROM 20130619 TO 20150322;REEL/FRAME:035632/0661