# US 6,720,147 B2 · Nanoparticles having oligonucleotides attached thereto and uses therefor

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- Number: US 6,720,147 B2
- Document ID: US6720147B2
- Status: Granted
- Kind: B2
- Application number: 09/976,577
- Filed: 2001-10-12
- Published: 2002-10-24
- Granted: 2004-04-13
- Earliest claimed: 1996-07-29
- Assignees: Nanosphere, Inc.
- Inventors: Chad A. Mirkin; Robert L. Letsinger; Robert C. Mucic; James J. Storhoff; Robert Elghanian; Thomas A. Taton
- Web page: https://patentgrep.com/patent/US6720147B2

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- B82Y 15/00: Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6839: Triple helix formation or other higher order conformations in hybridisation assays
- B01J 2219/00274: Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J 2219/005: Beads
- B01J 2219/00585: Parallel processes
- B01J 2219/00596: Solid-phase processes
- B01J 2219/00603: Making arrays on substantially continuous surfaces
- B01J 2219/00648: by the use of solid beads
- B01J 2219/00653: the compounds being bound to electrodes embedded in or on the solid supports
- B01J 2219/00659: Two-dimensional arrays
- B01J 2219/00702: Processes involving means for analysing and characterising the products
- B01J 2219/00722: Nucleotides
- B82B 1/00: Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
- C07B 2200/11: Compounds covalently bound to a solid support
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- Y10S 977/924: using nanostructure as support of dna analysis
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B82B: NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
- C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

The invention provides methods of detecting a nucleic acid. The methods comprise contacting the nucleic acid with one or more types of particles having oligonucleotides attached thereto. In one embodiment of the method, the oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change (preferably a color change) is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The invention also provides compositions and kits comprising particles. The invention further provides methods of synthesizing unique nanoparticle-oligonucleotide conjugates, the conjugates produced by the methods, and methods of using the conjugates. In addition, the invention provides nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. Finally, the invention provides a method of separating a selected nucleic acid from other nucleic acids.

## Claims

### Claim 1

Nanoparticle-oligonucleotide conjugates which are nanoparticles having oligonucleotides attached to them, the oligonucleotides being present on a surface of the nanoparticles at a surface density of at least 10 picomoles/cm2, at least some of the oligonucleotides having a sequence complementary to at least one portion of the sequence of a nucleic acid or another oligonucleotide.

### Claim 2 (depends on 1)

The conjugates of claim 1, wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of at least 15 picomoles/cm2.

### Claim 3 (depends on 2)

The conjugates of claim 2, wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of from about 15 picomoles/cm2 to about 40 picomoles/cm2.

### Claim 4 (depends on 1)

The conjugates of claim 1, wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles.

### Claim 5 (depends on 4)

The conjugates of claim 4, wherein the nanoparticles are gold nanoparticles.

### Claim 6 (depends on 1, 2, 3, 4, 5)

A kit comprising a container holding nanoparticle-oligonucleotide conjugates according to any one of claims 1, 2, 3, 4, 5.

### Claim 7 (depends on 1, 2, 3, 4, 5)

Nanomaterials or nanostructures composed of nanoparticle-oligonucleotide conjugates according to any one of claims 1, 2, 3, 4, 5, the nanoparticles being held together by oligonucleotide connectors.

### Claim 8

Nanoparticle-oligonucleotide conjugates which are nanoparticles having oligonucleotides attached to them, the oligonucleotides being present on the surface of the nanoparticles at a surface density sufficient so that the conjugates are stable, at least some of the oligonucleotides having a sequence complementary to at least one portion of the sequence of a nucleic acid or another oligonucleotides, wherein the oligonucleotides are attached to the nanoparticles in a stepwise ageing process comprising (i) contacting the oligonucleotides with the nanoparticles in a first aqueous solution for a period of time sufficient to allow some of the oligonucleotides to bind to the nanoparticles; (ii) adding at least one salt to the aqueous solution to create a second aqueous solution; and (iii) contacting the oligonucleotides and nanoparticles in the second aqueous solution for an additional period of time to enable additional oligonucleotides to bind to the nanoparticles.

### Claim 9 (depends on 8)

The conjugates of claim 8, wherein the salt solution has an ionic strength sufficient to overcome at least partially the electrostatic attraction or repulsion of the oligonucleotides for the nanoparticles and the electrostatic repulsion of the oligonucleotides for each other.

### Claim 10 (depends on 8)

The conjugates of claim 8 wherein the nanoparticles are metal nanoparticles or semiconductor nanoparticles.

### Claim 11 (depends on 10)

The conjugates of claim 10 wherein the nanoparticles are gold nanoparticles.

### Claim 12 (depends on 11)

The conjugates of claim 11 wherein the oligonucleotides include a moiety comprising a functional group which can bind to a nanoparticle.

### Claim 13 (depends on 8)

The conjugates of claim 8 wherein all of the salt is added to the water in a single addition.

### Claim 14 (depends on 8)

The conjugates of claim 8 wherein the salt is added gradually over time.

### Claim 15 (depends on 8)

The conjugates of claim 8 wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and a combination of two or more these salts in a phosphate buffer.

### Claim 16 (depends on 15)

The conjugates of claim 15 wherein the salt is sodium chloride in a phosphate buffer.

### Claim 17 (depends on 8)

The conjugates of claim 8 wherein the oligonucleotides are present on surface of the nanoparticles at a surface density of at least 10 picomoles/cm2.

### Claim 18 (depends on 12)

The conjugates of claim 12 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of at least 15 picomoles/cm2.

### Claim 19 (depends on 18)

The conjugates of claim 18 wherein the oligonucleotides are present on the surface of the nanoparticles at a surface density of from about 15 picomoles/cm2 to about 40 picomoles/cm2.