Biomolecule analysis by rolling circle amplification and SERS detection

• US 20040161750A1
• Filed: 02/14/2003
• Published: 08/19/2004
• Est. Priority Date: 02/14/2003
• Status: Active Grant

• Current Assignee: Intel Corporation
• Original Assignee: Intel Corporation
• Inventors: Su, Xing, Sun, Lei
• Granted Patent: US 7,192,703 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons

Abstract

The present methods, compositions and systems are concerned with biomolecule 130 detection, identification and/or quantification by rolling circle amplification (RCA) and Raman detection. In particular embodiments of the invention, the RCA is exponential RCA or linear RCA. In some embodiments of the invention, the Raman detection is SERS or SERRS. The circular DNA template 150, 210, 310 to be amplified may comprise one or more polythymidine 320 residues, resulting in amplification products 170, 230, 250, 330, 410 containing multiple polyadenylate 340, 420 residues. The polyadenylates 340, 420 may be directly detected by Raman detection. Alternatively, one or more Raman labels may be incorporated into the amplification products 170, 230, 250, 330, 410 to facilitate Raman detection. Because of the amplification produced by LRCA or ERCA and the enhanced Raman signal produced by multiple polyadenylates 340, 420 and/or Raman labels, detection of single copy biomolecules 130 is feasible using the disclosed methods, compositions and/or systems.

Claims

1. A method comprising:

a) attaching one or more target biomolecules to a substrate;

b) using a first primer to produce one or more rolling circle amplification (RCA) products;

c) detecting each amplification product by Raman spectroscopy; and

d) identifying one or more target biomolecules by the presence of one or more amplification products.

2. The method of claim 1, wherein each amplification product is attached to a target biomolecule.

3. The method of claim 1, wherein the target biomolecules are nucleic acids, polynucleotides or oligonucleotides.

4. The method of claim 3, wherein the nucleic acids are RNA or DNA.

5. The method of claim 3, wherein the first primer comprises the 3′

end of the target biomolecules.

6. The method of claim 3, wherein the first primer hybridizes to the target biomolecules.

7. The method of claim 1, wherein the target biomolecules are peptides, polypeptides or proteins.

8. The method of claim 7, wherein the target biomolecules bind to one or more ligands.

9. The method of claim 8, wherein the ligands are conjugated to said first primer.

10. The method of claim 9, wherein the ligands are antibodies, antibody fragments, single-chain antibodies, humanized antibodies or chimeric antibodies.

11. The method of claim 1, wherein the rolling circle amplification is linear RCA (LRCA) or exponential RCA (ERCA).

12. The method of claim 1, wherein the Raman spectroscopy is surface enhanced Raman spectroscopy (SERS) or surface enhanced resonance Raman spectroscopy (SERRS).

13. The method of claim 1, further comprising hybridizing a circular, single-stranded DNA template to said first primer, said template containing one or more polythymidine residues.

14. The method of claim 11, wherein the rolling circle amplification is ERCA and a second primer is added to the amplification mixture.

15. The method of claim 1, further comprising exposing the amplification products to a silver colloid solution.

16. The method of claim 1, further comprising generating metal nanoparticles by a reaction between aqueous metal salts and the amplification products.

17. The method of claim 16, further comprising treating the amplification products with an aldehyde to form a reducing agent.

18. The method of claim 16, wherein the metal salts comprise Ag⁺, Cu⁺, Cu²⁺, Au⁺, Au³⁺, Pd²⁺, Pd⁴⁺ or Al³⁺.

19. The method of claim 1, further comprising incorporating one or more Raman labels into said amplification products.

20. The method of claim 1, further comprising quantifying the amount of one or more target biomolecules.

21. A system for detecting biomolecules comprising:

a) one or more target biomolecules attached to a substrate;

b) one or more rolling circle amplification products associated with the target biomolecules; and

c) a Raman detector to detect said amplification products.

22. The system of claim 21, further comprising an excitatory light source to excite said amplification products.

23. The system of claim 22, wherein said light source comprises a laser.

24. The system of claim 21, wherein said Raman detector comprises one or more units selected from the group consisting of a CCD camera, an avalanche photodiode, a spectrophotometer, a photomultiplier tube, a spectrograph, a charged injection device, a photodiode array, an array detector and a phototransistor array.

25. The system of claim 21, further comprising metal nanoparticles.

26. The system of claim 25, wherein said nanoparticles comprise at least one metal selected from the group consisting of silver, gold, copper, platinum and aluminum.

27. The system of claim 21, further comprising one or more Raman labels attached to said amplification products.

28. The system of claim 21, further comprising a computer to analyze data from said Raman detector.

29. A composition comprising:

a) one or more target biomolecules;

b) one or more rolling circle amplification products associated with the target biomolecules; and

c) one or more nanoparticles adjacent to said amplification products.

30. The composition of claim 29, wherein said amplification products comprise one or more primers.

31. The composition of claim 29, further comprising a circular DNA template, said template comprising one or more polythymidine residues.

32. The composition of claim 29, further comprising one or more Raman labels attached to said amplification products through hybridization, intercalation or reaction with modified bases in the amplification products.