DNA microarray having hairpin probes tethered to nanostructured metal surface

• US 8,957,002 B2
• Filed: 11/05/2008
• Issued: 02/17/2015
• Est. Priority Date: 11/05/2007
• Status: Expired due to Fees

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Miller, Benjamin L., Krauss, Todd D., Rothberg, Lewis J., Peng, Hsin-I
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): Flinders, Jeremy C
• Application Number:
  US12/265,047
• Publication Number:
  US 20090137418A1
• Time in Patent Office: 2,295 Days
• Field of Search: 506/9
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• 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: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support
• CPC Class Codes: C40B 60/08 Integrated apparatus specially adapted for both creating and screening libraries
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

A sensor chip and detection device are disclosed. The sensor chip includes a substrate, at least a portion of which is covered by a metal nanoparticle film; a first nucleic acid molecule that is characterized by being able to (i) self-anneal into a hairpin conformation and (ii) hybridize specifically to a target nucleic acid molecule, the first nucleic acid molecule having first and second ends, which first end is tethered to the metal nanoparticle film; and a first fluorophore bound to the second end of the first nucleic molecule. When the first nucleic acid molecule is in the hairpin conformation, the metal nanoparticle film substantially quenches fluorescent emissions by the first fluorophore, and when the first nucleic acid molecule is in a non-hairpin conformation fluorescent emissions by the first fluorophore are surface plasmon-enhanced.

Claims

1. A sensor chip comprising:

a substrate at least a portion of which is covered by a metal nanoparticle film, wherein the metal nanoparticle film comprises nanoparticles covalently attached to the substrate, the nanoparticles having a size in the range of 3.6 to 31.4 nm in diameter, and the metal nanoparticle film having a surface roughness of between about 0.7 nm and about 3 nm;

a first nucleic acid molecule that is characterized by being able to (i) self-anneal into a hairpin conformation and (ii) hybridize specifically to a target nucleic acid molecule, the first nucleic acid molecule having first and second ends, which first end is tethered to the metal nanoparticle film; and

a fluorophore bound to the second end of the first nucleic acid molecule;

whereby when the first nucleic acid molecule is in the hairpin conformation, the metal nanoparticle film substantially quenches fluorescent emissions by the fluorophore, and when the first nucleic acid molecule is in a non-hairpin conformation fluorescent emissions by the fluorophore are surface plasmon-enhanced and yield an R-value of at least about 3.4.

2. The sensor chip according to claim 1 wherein the metal nanoparticle film comprises a surface roughness of between about 1 nm and about 2.4 nm.

3. The sensor chip according to claim 1 wherein the metal nanoparticle film comprises a surface roughness of between about 1 nm and about 2 nm.

4. The sensor chip according to claim 1 wherein the metal nanoparticle film is translucent.

5. The sensor chip according to claim 4 wherein the metal nanoparticle film is less than about 50 nm thick.

6. The sensor chip according to claim 1 wherein the metal nanoparticle film comprises gold, silver, platinum, titanium, copper, gallium, or aluminum.

7. The sensor chip according to claim 1 wherein the first nucleic acid molecule is less than about 60 nucleotides in length.

8. The sensor chip according to claim 1 wherein the fluorophore is a dye, a protein, or a semiconductor nanocrystal.

9. The sensor chip according to claim 1 wherein the substrate is light transmissive.

10. The sensor chip according to claim 1 wherein the substrate is capable of quenching fluorescence of the fluorophore.

11. The sensor chip according to claim 1 wherein the substrate is a metal and is different from the metal used for the metal nanoparticle film.

12. The sensor chip according to claim 11 wherein the metal nanoparticle film comprises silver nanoparticles and the substrate comprises gold, platinum, titanium, copper, gallium, or aluminum.

13. The sensor chip according to claim 1 wherein the sensor chip comprises:

a plurality of nucleic acid molecules that are different from one another and are characterized by being able to (i) self-anneal into a hairpin conformation and (ii) hybridize specifically to a target nucleic acid molecule, each of the plurality of nucleic acid molecules having first and second ends, which first end is tethered to the metal nanoparticle film; and

a fluorophore bound to the second end of each of the plurality of nucleic acid molecules;

whereby when the nucleic acid molecules are in the hairpin conformation, the metal nanoparticle film substantially quenches fluorescent emissions by the fluorophore bound thereto, and when the nucleic acid molecules are in a non-hairpin conformation fluorescent emissions by the fluorophore bound thereto are surface plasmon-enhanced and yield an R-value of at least about 3.4.

14. The sensor chip according to claim 13, wherein the plurality of nucleic acid molecules are present in the form of an array.

15. A biological sensor device comprising:

a sensor chip according to claim 1;

a light source that illuminates the sensor chip at a wavelength suitable to induce fluorescent emissions by the fluorophore(s); and

a detector positioned to detect fluorescent emissions by the fluorophore(s).

16. The biological sensor device according to claim 15, wherein the sensor chip is positioned in a flow cell with the chip surface comprising the portion covered by the metal nanoparticle film exposed to the interior of the flow cell.

17. The biological sensor device according to claim 16 wherein the metal nanoparticle film is light transmissive, and the light source is positioned to illuminate the sensor chip from a side thereof that is opposite the chip surface comprising the portion covered by the metal nanoparticle film.

18. The biological sensor device according to claim 16 wherein the metal nanoparticle film is light transmissive, and the detector is positioned to detect fluorescent emissions emanating from the chip surface comprising the portion covered by the metal nanoparticle film.

19. A method of detecting the presence of a target nucleic acid molecule in a sample comprising:

exposing the sensor chip according to claim 1 to a sample under conditions effective to allow any target nucleic acid molecule in the sample to hybridize to the nucleic acid molecules, causing the nucleic acid molecules to adopt the non-hairpin conformation;

illuminating the sensor chip with light sufficient to cause emission of fluorescence by the fluorophore(s); and

determining whether or not the sensor chip emits fluorescent emissions of one or more of the fluorophores upon said illuminating, wherein fluorescent emission by the sensor chip indicates that a nucleic acid molecule is in the non-hairpin conformation and therefore that its target nucleic acid molecule is present in the sample.

20. The method according to claim 19 wherein the target nucleic acid molecule is specific for a pathogen, a disease state, a genetic marker, or a forensic target, or associated with a breeding trait for a plant or animal.

21. The sensor chip according to claim 1 wherein the metal nanoparticle film comprises a nanoparticle density of between 315 to 722 nanoparticles per μ

m² as measured by atomic force microscopy.

22. The sensor chip according to claim 1 wherein the metal nanoparticle film has an average particle size of about 4.9 nm to about 6.93 nm.

23. The sensor chip according to claim 1 wherein the sensor comprises an R-value of at least about 6.

24. The sensor chip according to claim 1 wherein the nanoparticles are covalently attached to the substrate via a linker molecule.