Nanoparticles having oligonucleotides attached thereto and uses therefor

• US 20090325812A1
• Filed: 02/02/2007
• Published: 12/31/2009
• Est. Priority Date: 07/29/1996
• Status: Active Grant

• Current Assignee: Nanosphere Incorporated (DiaSorin S.p.A.), Northwestern University
• Original Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Inventors: Taton, Thomas A., Letsinger, Robert L., Mucic, Robert C., Mirkin, Chad A., Storhoff, James J., Elghanian, Robert
• Granted Patent: US 8,323,888 B2
• Time in Patent Office: Days
• US Class Current: 506/8
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00603 Making arrays on substantially continuous surfaces
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82B 1/00 Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: 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
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6839 : Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2525/161 : incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 : incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2537/125 : Sandwich assay format
• CPC Class Codes: C12Q 2537/143 : Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/143 : Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 : Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/625 : being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: 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
• CPC Class Codes: Y10S 977/924 : using nanostructure as support of dna analysis

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

1-432. -432. (canceled)

433. A method for the identification and/or the quantification of a target compound obtained from a sample, comprising the steps of:

putting into contact the target compound with a capture molecule in order to allow a specific binding between said target compound with said capture molecule, said capture molecule being fixed upon a surface of a solid support according to an array comprising discrete regions with a density of at least 20 of said discrete regions per cm², each of said discrete regions being fixed with one species of capture molecules;

performing a catalytic reduction of a metal present in solution leading to formation of a metallic precipitate in one or more of said discrete regions;

determining the possible presence and quantification of precipitate(s) in said discrete region(s); and

correlating the presence and quantification of the precipitate(s) at the discrete region(s) with the identification and/or a quantification of said target compound.

434. The method according to claim 433, wherein said metallic precipitate is a magnetic metallic compound.

435. The method according to claim 433, wherein the catalytic reduction leading to the formation of the metallic precipitate is a reduction of a metal in the presence of an enzyme.

436. The method according to claim 433, wherein the catalytic reduction leading to the formation of the metallic precipitate is a chemical reduction of silver in the presence of colloidal gold particles coupled to the bound target compound.

437. The method according to claim 433, wherein the specific binding between the target compound and its corresponding capture molecule is a hybridization between two nucleotide sequences.

438. The method according to claim 433, wherein the binding between the target compound and its corresponding capture molecule is a reaction between an antigenic structure and its corresponding antibody or a hypervariable portion thereof.

439. The method according to claim 433, wherein the binding between the target compound and its corresponding capture molecule is a reaction between a receptor and its corresponding ligand.

440. The method according to claim 433, wherein the possible presence and quantification of a precipitate is obtained by reflection, absorption or diffusion of a light beam upon said precipitate.

441. The method of claim 440, wherein the light beam is a laser beam.

442. The method according to claim 433, wherein the presence and quantification of a precipitate in a discrete region is obtained by variation of an electromagnetic field or the conductance of an electric current.

443. The method according to claim 433, for the quantification of volume of one or more precipitate(s) upon a defined surface of the solid support, wherein images of said defined surface containing one or more precipitate(s) and corresponding to different views, said images containing analogue information, are taken by one or more camera(s) upon illumination by one or more illuminant source(s), spatially arranged relatively to each other according to a predetermined pattern and wherein the corresponding image analogue information of said defined surface containing said precipitate(s) are transformed and converted into digital form or a set of digital forms and compared to a first and to a second reference standards to determine the volume of the precipitate(s) to be quantified.

444. The method according to claim 443, wherein the first reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support without precipitate.

445. The method according to claim 444, wherein the second reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support containing precipitate(s) of known volume.

446. The method of claim 433, wherein said metallic precipitate is formed on the surface of a particle associated with said target compound.

447. The method of claim 433, wherein said sample is a biological sample.

463. A computer comprising program code stored thereon for performing the steps of determining the possible presence and quantification of a precipitate in discrete regions and correlating the presence and quantification of said precipitate at the discrete regions with the identification and/or the quantification of a target compound, according to the method of claim 433, when said program code is run on said computer.

464. A computer program product comprising program code means stored on a computer readable medium for performing the steps of determining the possible presence and quantification of a precipitate in a discrete region and correlating the presence and quantification of the precipitate at the discrete region with the identification and/or the quantification of a target compound, according to the method of claim 433, when said program is run on a computer.

448. A diagnostic and/or quantification apparatus comprising:

a solid support comprising an array comprising at least 20 discrete regions per cm² comprising multiple species of capture molecules, each of said discrete regions being fixed with one species of capture molecule which recognizes a target compound, wherein binding of said target compound to one or more of said multiple species of capture molecules leads to a catalytic reduction of a metal present in solution, and a formation of a metallic precipitate in one or more of said discrete region(s);

a detection and quantification device for detecting and quantifying said precipitate in said discrete region(s); and

a computer programmed to collect the results obtained from said detection and quantification device.

449. The apparatus according to claim 448, comprising one or more sensor(s) provided with camera(s) and with one or more illuminant source(s) which are spatially arranged relatively to each other according to a predetermined pattern and which are associated with an analogue information acquisition system, said information being measured by using sensor(s) and being converted into digital form by a processing unit.

450. The apparatus according to claim 449, wherein the camera(s) are CCD or CMOS camera(s).

451. The apparatus according to claim 449, wherein the illuminant source is an infra-red light having a wavelength similar to a metal crystal contained in the precipitate(s).

452. The apparatus according to claim 449, which comprises a set of illuminant sources regularly spaced from each other in a plane, each of said sources corresponding to a light spot being automatically switched on, simultaneously or successively.

453. The apparatus according to claim 449, which comprises one camera and one illuminant source placed above the solid support, said camera and illuminant source being movable in three dimensions in space.

454. The apparatus according to claim 449, which comprises two or more cameras oppositely arranged in a plane and placed above the solid support, the apparatus comprising further one or more illuminant source(s) placed under the solid support.

455. The apparatus according to claim 449, which comprises three or more cameras arranged according to a triangular plane or another regular or irregular pattern and placed above the solid support and further comprising one or more illuminant source(s) placed under the solid support.

456. The apparatus according to claim 449, which comprises, placed above the solid support, one camera and a first illuminant source and, under said camera, a second illuminant source placed under the solid support, the two illuminant sources being placed almost symmetrically according to the position of the solid support.

457. The apparatus of claim 448, wherein the information(s) recorded upon said solid support are barcodes.

458. The apparatus of claim 448, wherein the results are the formation of a precipitate at a specific location.

459. The apparatus of claim 448, further comprising a device for reading information recorded upon said solid support.

460. The apparatus of claim 459, wherein said device for reading information comprises a bar code reader.

461. The apparatus of claim 448 wherein said computer is programmed to recognize discrete regions bearing said capture molecules.

462. The apparatus of claim 448, wherein said computer is programmed to detect and/or quantitate said target compounds.

465. A method for the identification and/or quantification of an analyte nucleic acid in a sample, comprising the steps of:

(a) contacting a sample suspected of containing said analyte nucleic acid with a substrate comprising one or more oligonucleotide molecules attached to said substrate wherein said one or more oligonucleotide molecules comprise a sequence that specifically binds said analyte nucleic acid when present,

wherein said analyte nucleic acid comprises a first portion complementary to said one or more oligonucleotide molecules attached to said substrate;

wherein said one or more oligonucleotide molecules are fixed upon a surface of said substrate in an array of regions, each region containing a different oligonucleotide;

(b) contacting said substrate with an oligonucleotide conjugated to a nanoparticle, wherein said oligonucleotide that is conjugated to a nanoparticle is complementary to a second portion of said analyte nucleic acid, and wherein binding of said oligonucleotide conjugated to a nanoparticle to said analyte nucleic acid forms a modified colloid;

(c) staining said modified colloid with silver to form a metallic precipitate where said modified colloid hybridized to bound analyte nucleic acid is present;

(d) determining the possible presence and quantity of said metallic precipitate; and

(e) correlating the presence and quantity of the metallic precipitate with the identification and/or quantification of said analyte nucleic acid.

466. The method according to claim 465, wherein said nanoparticle is comprised of gold, platinum or a magnetic material.

467. The method according to claim 465, wherein said nanoparticle is gold.

468. The method according to claim 465, wherein said analyte nucleic acid has no mismatches with at least one of said oligonucleotide molecules attached to said substrate or said oligonucleotide conjugated to a nanoparticle.

469. The method of claim 465, wherein determining the possible presence of said metallic precipitate is conducted by observing a detectable change.

470. The method of claim 469, wherein said substrate is a transparent substrate and said detectable change is the formation dark areas due to the absorption of light and said determining is conducted by observing said absorption.

471. The method of any one of claims 465 to 467, wherein said staining forms said metallic precipitate on said nanoparticles.

472. The method of claim 465, where in said array comprises a row of spots on said substrate.

473. The method of claim 465, wherein said analyte nucleic acid is from a biological source.

516. A computer comprising program code stored thereon for performing the steps of determining the possible presence and quantification of a precipitate in discrete regions and correlating the presence and quantification of said precipitate at the discrete regions with the identification and/or the quantification of a target compound, according to the method of claim 465, when said program code is run on said computer.

517. A computer program product comprising program code means stored on a computer readable medium for performing the steps of determining the possible presence and quantification of a precipitate in a discrete region and correlating the presence and quantification of the precipitate at the discrete region with the identification and/or the quantification of a target compound, according to the method of claim 465, when said program is run on a computer.

474. A method for identifying and/or quantifying a nucleic acid in a sample, comprising the steps of:

(a) contacting said sample with one or more oligonucleotide molecules attached to a substrate to allow specific binding between said nucleic acid and said one or more oligonucleotide molecules to form a nucleic acid hybridized to said substrate,

wherein said nucleic acid comprises a first portion complementary to at least one of said one or more oligonucleotide molecules attached to said substrate and a second portion complementary to a oligonucleotide that is conjugated to a nanoparticle;

wherein said one or more oligonucleotide molecules attached to said substrate are fixed upon a surface of said substrate in an array of regions, wherein each region contains a different oligonucleotide;

(b) contacting said nucleic acid hybridized to said substrate with a nanoparticle conjugated to an oligonucleotide that is complementary to said second portion of said nucleic acid;

(c) detecting the presence and quantity of said nucleic acid by reducing silver present in solution, wherein a metallic precipitate is formed in one or more of said regions when said nucleic acid is present; and

(d) correlating the presence and quantity of the precipitate(s) at the region(s) with the identifying and/or quantifying of said nucleic acid.

475. The method according to claim 474, wherein said nanoparticle is comprised of gold, platinum or a magnetic material.

476. The method according to claim 474, wherein said nanoparticle is gold.

477. The method according to claim 474, wherein said nucleic acid has no mismatches with at least one of said oligonucleotide molecules attached to said substrate or said oligonucleotide conjugated to a nanoparticle.

478. The method of claim 474, wherein determining the possible presence of said metallic precipitate is conducted by observing a detectable change.

479. The method of claim 478, wherein said substrate is a transparent substrate, said detectable change is the formation dark areas due to the absorption or scattering of light, and said determining is conducted by observing said absorption or scattering of light.

480. The method of claim 478, wherein said detectable change is observed by an electrochemical technique.

481. The method of claim 480, wherein said electrochemical technique is cyclic voltammetry.

482. The method of any one of claims 474 to 481, wherein said metallic precipitate forms on said nanoparticle.

483. The method of claim 474, where in said array comprises a plurality of oligonucleotide molecules attached to said substrate.

484. The method of claim 474, wherein said nucleic acid is from a biological source.

485. The method of claim 474, wherein said nucleic acid is from a cell sample, tissue sample, or a biological fluid.

486. An apparatus comprising:

a solid surface comprising an array of one or more oligonucleotides attached to said surface, and at least one nucleic acid having a first portion and a second portion, wherein said first portion is hybridized to said oligonucleotide attached to said surface;

wherein said apparatus further comprises a nanoparticle conjugated to an oligonucleotide hybridized to said second portion;

wherein when said apparatus is contacted with a silver stain solution, silver is reduced to form a metallic precipitate where said nanoparticle conjugated to an oligonucleotide is located;

and wherein said apparatus further comprises a scanning apparatus for scanning the said solid surface to detect or quantitate said metallic precipitate, wherein said apparatus is linked to a computer loaded with software for calculating grayscale measurements obtained from said scanning apparatus.

487. The apparatus of claim 486, wherein said scanning apparatus is a flat bed scanner.

488. The apparatus of claim 486 or claim 487, wherein said computer is programmed to quantitate the nucleic acid present.

489. The apparatus of claim 487, wherein said apparatus comprises a set of illumination sources arranged in a row that are switched on simultaneously.

490. The apparatus of claim 490, wherein said apparatus quantitates the concentration of nucleic acid present in a sample based upon the nucleic acid concentration of standards.

491. A method for the identification and/or the quantification of a target compound obtained from a sample, comprising the steps of:

putting into contact the target compound with a capture molecule in order to allow a specific binding between said target compound with said capture molecule, said capture molecule being fixed upon a surface of a solid support according to an array comprising rows of discrete regions, each of said discrete regions being fixed with one species of capture molecules;

performing a catalytic reduction of a metal present in solution leading to formation of a metallic precipitate in one or more of said discrete regions;

determining the possible presence and quantification of precipitate(s) in said discrete region(s); and

correlating the presence and quantification of the precipitate(s) at the discrete region(s) with the identification and/or a quantification of said target compound.

492. The method according to claim 491, wherein said metallic precipitate is a magnetic metallic compound.

493. The method according to claim 491, wherein the catalytic reduction leading to the formation of the metallic precipitate is a chemical reduction of silver in the presence of colloidal gold particles coupled to the bound target compound.

494. The method according to claim 491, wherein the specific binding between the target compound and its corresponding capture molecule is a hybridization between two nucleotide sequences.

495. The method according to claim 491, wherein the possible presence and quantification of a precipitate is obtained by reflection, absorption or diffusion of a light beam upon said precipitate.

496. The method according to claim 495, wherein the possible presence and quantification of a precipitate is obtained by absorption.

497. The method according to claim 491, wherein the presence and quantification of a precipitate in a discrete region is obtained by variation of an electromagnetic field or the conductance of an electric current.

498. The method of claim 497, wherein the conductance of electric current is determined by cyclic voltammetry.

499. The method according to claim 491, for the quantification of volume of one or more precipitate(s) upon a defined surface of the solid support, wherein images of said defined surface containing one or more precipitate(s) and corresponding to different views, said images containing analogue information, are taken by one or more camera(s) upon illumination by one or more illuminant source(s), spatially arranged relatively to each other according to a predetermined pattern and wherein the corresponding image analogue information of said defined surface containing said precipitate(s) are transformed and converted into digital form or a set of digital forms and compared to a first and to a second reference standards to determine the volume of the precipitate(s) to be quantified.

500. The method according to claim 499, wherein the image is provided by a scanner.

501. The method according to claim 500, wherein the first reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support without precipitate.

502. The method according to claim 500, wherein the second reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support containing precipitate(s) of known volume or amount.

503. The method according to claim 500, wherein the second reference standard corresponds to a digital form or a set of digital forms obtained from analogue information contained in images taken on the surface of said solid support containing precipitate(s) of known amount.

504. The method of claim 491, wherein said metallic precipitate is formed on the surface of a particle associated with said target compound.

505. The method of claim 491, wherein said sample is a biological sample.

513. The apparatus of claim 503, wherein said computer is programmed to detect and/or quantitate said target compounds.

514. A computer comprising program code stored thereon for performing the steps of determining the possible presence and quantification of a precipitate in discrete regions and correlating the presence and quantification of said precipitate at the discrete regions with the identification and/or the quantification of a target compound, according to the method of claim 491, when said program code is run on said computer.

515. A computer program product comprising program code means stored on a computer readable medium for performing the steps of determining the possible presence and quantification of a precipitate in a discrete region and correlating the presence and quantification of the precipitate at the discrete region with the identification and/or the quantification of a target compound, according to the method of claim 491, when said program is run on a computer.

506. A diagnostic and/or quantification apparatus comprising:

a solid support comprising an array comprising rows of discrete regions comprising multiple species of capture molecules, each of said discrete regions being fixed with one species of capture molecule which recognizes a target compound, wherein binding of said target compound to one or more of said multiple species of capture molecules leads to a catalytic reduction of a metal present in solution, and a formation of a metallic precipitate in one or more of said discrete region(s);

a detection and quantification device for detecting and quantifying said precipitate in said discrete region(s); and

a computer programmed to collect the results obtained from said detection and quantification device.

507. The apparatus according to claim 506, wherein the detection and quantification device is a spectrophotometer.

508. The apparatus according to claim 506, wherein the detection and quantification device is a scanner.

509. The apparatus according to claim 506, comprising one or more sensor(s) provided with camera(s) and with one or more illuminant source(s) which are spatially arranged relatively to each other according to a predetermined pattern and which are associated with an analogue information acquisition system, said information being measured by using sensor(s) and being converted into digital form by a processing unit.

510. The apparatus according to claim 509, which comprises one camera and one illuminant source placed below the solid support.

511. The apparatus of claim 506, wherein the results are the formation of a precipitate at a specific location.

512. The apparatus of claim 506 wherein said computer is programmed to recognize discrete regions bearing said capture molecules.

508-1. The apparatus according to claim 506, wherein the camera is part of a scanner.

509-2. The apparatus according to claim 506, which comprises a set of illuminant sources regularly spaced from each other in a plane, each of said sources corresponding to a light spot being automatically switched on, simultaneously or successively.