Methods and device for DNA sequencing using surface enhanced raman scattering (SERS)

• US 7,476,501 B2
• Filed: 03/26/2002
• Issued: 01/13/2009
• Est. Priority Date: 03/26/2002
• Status: Expired due to Fees

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing, Koo, Tae-Woong, Chan, Selena
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Wilder; Cynthia B
• Application Number:
  US10/108,128
• Publication Number:
  US 20030187237A1
• Time in Patent Office: 2,485 Days
• Field of Search: 435/6, 435/287.2, 536/23.1, 204/164
• US Class Current: 435/6.1
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/60 Detection means characterised by use of a special device
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: G01N 2021/653 Coherent methods [CARS]
• CPC Class Codes: G01N 2021/656 Raman microprobe
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons
• CPC Class Codes: G01N 33/587 Nanoparticles

Abstract

The methods and apparatus disclosed herein concern nucleic acid sequencing by enhanced Raman spectroscopy. In certain embodiments of the invention, exonuclease treatment of the nucleic acids 109 results in the release of nucleotides 110. The nucleotides may pass from a reaction chamber 101 through a microfluidic channel 102 and enter a nanochannel or microchannel 103. The nanochannel or microchannel 103 may be packed with nanoparticle 111 aggregates containing hot spots for Raman detection. As the nucleotides 110 pass through the nanoparticle 111 hot spots, they may be detected by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS). Identification of the sequence of nucleotides 110 released from the nucleic acid 109 provides the nucleic acid sequence. Other embodiments of the invention concern apparatus 100 for nucleic acid sequencing.

Claims

1. A method of identifying a single nucleotide by Raman spectroscopy, comprising:

contacting a nucleic acid molecule attached at one end to a immobilization surface with one or more exonucleases to remove the single nucleotide from an unattached end of the nucleic acid molecule;

separating the single nucleotide from the immobilized nucleic acid,

transferring the single nucleotide to a hot spot comprising a plurality of cross-linked nanoparticle aggregates packed within a microfluidic channel, wherein association of the single nucleotide with the hot spot enhances Raman signaling; and

identifying the single nucleotide by Raman spectroscopy.

2. The method of claim 1, wherein the single nucleotide is removed from the nucleic acid by exonuclease activity.

3. The method of claim 1, wherein the plurality of nanoparticle aggregates comprises between two to six nanoparticles per aggregate.

4. The method of claim 1, wherein said nucleic acid is immobilized in a reaction chamber.

5. The method of claim 4, wherein a single nucleic acid is immobilized in said reaction chamber.

6. The method of claim 1, wherein said the single nucleotide is identified by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS).

7. The method of claim 1, wherein said channel is a nanochannel or microchannel.

8. The method of claim 3, wherein the plurality of nanoparticle aggregates comprises two nanoparticles per aggregate.

9. The method of claim 3, wherein the nanoparticles comprise gold and/or silver, said nanoparticles between about 1 nm and 2 μ

m in size.

10. The method of claim 9, wherein the size of said nanoparticles is selected from the group consisting of about 10 to 50 nm, about 50 to 100 nm, about 10 to 100 nm, about 100 nm and about 200 nm.

11. The method of claim 8, wherein the surfaces of said nanoparticles are covalently modified with organic compounds.

12. The method of claim 1, further comprising:

attaching the nucleic acid molecule to the immobilization surface.

13. The method of claim 1, further comprising imposing an electric field to move the single nucleotide through said channel.

14. The method of claim 1, further comprising recording the time at which the single nucleotide passes through said channel.

15. The method of claim 1, wherein the single nucleotide produces a unique Raman signal.

16. The method of claim 1, wherein at least a portion of the nucleic acid molecule comprises a single stranded nucleic acid.

17. The method of claim 1, wherein the single nucleotide is a single unlabeled nucleotide.

18. The method of claim 1, wherein the single nucleotide is a single Raman labeled nucleotide.