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

• US 20030187237A1
• Filed: 03/26/2002
• Published: 10/02/2003
• Est. Priority Date: 03/26/2002
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing, Chan, Selena
• Granted Patent: US 7,476,501 B2
• Time in Patent Office: Days
• US Class Current: 536/22.1
• 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 comprising:

a) sequentially removing nucleotides from one end of at least one nucleic acid;

b) moving the nucleotides through a channel packed with nanoparticles;

c) identifying the nucleotides by Raman spectroscopy; and

d) determining the sequence of the nucleic acid.

2. The method of claim 1, wherein the nucleotides are removed from the nucleic acid by exonuclease activity.

3. The method of claim 1, further comprising attaching said nucleic acid to a surface.

4. The method of claim 3, 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 nucleotides are 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 1, wherein at least two nanoparticles are cross-linked together.

9. The method of claim 1, 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. A method comprising:

a) preparing a nucleic acid comprising labeled nucleotides;

b) sequentially removing nucleotides from one end of the nucleic acid;

c) moving the nucleotides through a channel packed with nanoparticles;

d) identifying the nucleotides by Raman spectroscopy; and

e) determining the sequence of the nucleic acid.

13. The method of claim 12, wherein said nucleotides are labeled with one or more Raman labels.

14. The method of claim 13, wherein each type of nucleotide is labeled with a distinguishable Raman label.

15. The method of claim 13, wherein only pyrimidine nucleotides are labeled.

16. The method of claim 12, further comprising separating said nucleotides from said nucleic acid.

17. The method of claim 12, further comprising imposing an electric field to move said nucleotides through said channel.

18. The method of claim 12, further comprising recording the time at which each nucleotide passes through said channel.

19. The method of claim 13, wherein each type of nucleotide produces a unique Raman signal.

20. An apparatus comprising:

a) a reaction chamber;

b) a first channel in fluid communication with said reaction chamber;

c) a second channel in fluid communication with said first channel;

d) a multiplicity of nanoparticles in said second channel; and

e) a Raman detector operably coupled to said second channel.

21. The apparatus of claim 20, wherein said Raman detector comprises a laser, a CCD camera and/or a photodiode array.

22. The apparatus of claim 21, wherein said CCD camera and/or photodiode array is operably coupled to a data processing unit.

23. The apparatus of claim 22, wherein said data processing unit comprises a computer.

24. The apparatus of claim 20, further comprising a first electrode and a second electrode, said electrodes to move nucleotides from said first channel into said second channel.

25. The apparatus of claim 20, wherein said first channel is a microfluidic channel.

26. The apparatus of claim 20, wherein said second channel is a nanochannel or a microchannel.

27. The apparatus of claim 20, wherein said nanoparticles are cross-linked together.

28. The apparatus of claim 27, wherein said cross-linked nanoparticles provide an enhanced Raman signal.

29. The apparatus of claim 20, said detector to detect nucleotides by surface enhanced Raman spectroscopy (SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Raman spectroscopy (CARS).