Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-11
2003-08-05
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S024300, C436S073000, C436S080000, C436S164000
Reexamination Certificate
active
06602669
ABSTRACT:
FIELD OF THE INVENTION
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
(a) Gold Nanoparticle Probes
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)].
(b) Silver Enhancement of Signal
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.
(c) Oligo- and Polynucleotide Arrays
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 calorimetric 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.”
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.
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,
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
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.
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
Garimella Viswanadham
Letsinger Robert L.
Horlick Kenneth R.
McDonnell & Boehnen Hulbert & Berghoff
Northwestern University
Strzelecka Teresa E
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