Simultaneous amplification and detection of ribonucleic acid be an optical method using surface plasmon resonance

• US 20080131939A1
• Filed: 05/30/2007
• Published: 06/05/2008
• Est. Priority Date: 05/30/2006
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Roper, D. Keith
• Granted Patent: US 7,998,672 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/628 being a surface plasmon resonance spectrometer
• CPC Class Codes: G01N 21/554 detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
• 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
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/587 Nanoparticles

Abstract

Methods of performing PCR are provided. Methods may include using an optical source to provide heating for thermocyling the PCR reaction. Methods may include using surface plasmon resonance and/or fluorescence resonance enhanced transfer to allow real-time monitoring of a PCR reaction. Methods may include immobilizing a template, primer, or polymerase on a surface such as a gold or other surface plasmon resonance active surface.

Claims

1. A method of performing a Polymerase-Chain-Reaction (PCR), the method comprising:

providing an aqueous medium comprising or in fluid contact with a template, a primer capable of annealing to the template, and a thermostable polymerase;

allowing the primer to anneal to the template;

heating, with an optical source, a portion of the aqueous medium to a temperature sufficient to allow elongation of the primer;

elongating the primer using the thermostable polymerase; and

heating, with an optical source, a portion of the aqueous medium to a temperature sufficient to denature of the elongated primer and the template.

2. The method of claim 1, wherein the aqueous solution is in fluid contact with a gold surface.

3. The method of claim 2, wherein the gold surface is the surface of a nanoparticle.

4. The method of claim 3, wherein the nanoparticle is selected from the group consisting of gold nanoparticles, Au—

Si nanoshells, and gold nanocylinders.

5. The method according to claim 2, further comprising immobilizing at least one of the template, the primer capable of annealing to the template, and the thermostable polymerase on the gold surface.

6. The method according to claim 5, wherein immobilizing at least one of the template, the primer capable of annealing to the template, and the thermostable polymerase on the gold surface comprises using N-hydroxysuccinimide.

7. The method according to claim 1, wherein the Polymerase-Chain-Reaction is a multiplex Polymerase-Chain-Reaction.

8. The method according to claim 1 wherein the heating, with an optical source, a portion of the aqueous medium to a temperature sufficient to allow elongation of the primer is performed on a millisecond time scale.

9. The method according to claim 1 wherein the heating, with an optical source, a portion of the aqueous medium to a temperature sufficient to allow denaturation of the elongated primer and the template is performed on a millisecond time scale.

10. The method according to claim 1, further comprising monitoring the elongation of the primer by measuring fluorescence resonance enhanced transfer.

11. The method according to claim 2, wherein the gold surface is a surface-plasmon-resonance active surface.

12. The method according to claim 11, further comprising detecting mass elongating of the primer and amplification of the template using surface plasmon resonance.

13. The method according to claim 1, wherein the optical source is capable of emitting light with a wavelength of about 500 nm to about 900 nm.

14. The method according to claim 1, wherein the optical source is selected from the group consisting of a laser, Ti:

sapphire laser, an argon-ion laser, a krypton laser, a xenon lamp, and an light-emitting diode.

16. The method according to claim 1 further comprising measuring amplification efficiencies by measuring fluorescence resonance enhanced transfer.

15. A method of real time detection of Polymerase-Chain-Reaction (PCR) amplicons by Surface-Plasmon-Resonance (SPR), said method comprising:

immobilizing a polymerase or a polynucleotide primer on a gold surface;

providing, in fluid contact with the gold surface, a PCR template, dNTPs, and polymerase or a polynucleotide primer if not already immobilized on the gold surface;

cycling the temperature of an area adjacent the gold surface so as to perform PCR using the polymerase, primers, PCR template, and dNTPs;

wherein cycling the temperature of an area adjacent the gold surface so as to perform PCR comprises heating the area adjacent the gold surface with an optical source; and

measuring rates and magnitudes of interaction between the template and molecules immobilized on the gold surface by analyzing mass-induced changes at resonance absorption with SPR.

17. The method according to claim 15, wherein cycling between the annealing temperature and elongation temperature occurs on a millisecond time scale.

18. The method according to claim 15, wherein cycling between the elongation temperature and elongation temperature occurs on a millisecond time scale.

19. The method according to claim 15 wherein the optical source stimulates surface plasmon resonance.

20. The method according to claim 19, wherein the optical source is capable of emitting light with a wavelength of about 500 nm to about 900 nm.

21. The method according to claim 19, wherein the optical source is selected from the group consisting of a laser, Ti:

sapphire laser, an argon-ion laser, a krypton laser, a xenon lamp, and an light-emitting diode.

22. The method according to claim 15, wherein the gold surface is on a nanoparticle.

23. In a method of conducting a polymerase chain reaction (PCR) of the type involving detecting a polynucleotide using PCR cycling rates, the improvement comprising:

inducing thermal dissipation with surface plasmon resonance in a sample believed to contain said polynucleotide while, simultaneously, detecting in said sample, without labels, said polynucleotide also by surface plasmon resonance so as to attain millisecond PCR cycling rates

24. The method according to claim 23, wherein the sample is femtomolar-sized.

25. The method according to claim 23, characterized by real-time femtomolar detection sensitivity.

26. The method according to claim 23, wherein said PCR is conducted as part of highly parallel PCR.

27. The method according to claims 23, wherein said polynucleotide is selected from the group consisting of DNA, RNA, polynucleic acid (PNA), and combinations of any thereof.

28. The method according to claim 23, further comprising heating said sample with a non-optical thermal source.