HYBRIDIZATION-BASED BIOSENSOR CONTAINING HAIRPIN PROBES AND USE THEREOF

• US 20090047670A1
• Filed: 08/14/2007
• Published: 02/19/2009
• Est. Priority Date: 08/14/2007
• Status: Active Grant

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: Miller, Benjamin L., Strohsahl, Christopher M.
• Granted Patent: US 7,811,764 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface 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 fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

Claims

1. An isolated nucleic acid molecule that is less than about 60 nucleotides in length, is characterized by being able to self-anneal into a hairpin conformation, and hybridizes over substantially the entire length thereof to nucleic acids from methicillin-resistant Staphylococcus spp.

2. The isolated nucleic acid molecule according to claim 1 further comprising a fluorophore tethered to one end thereof.

3. The isolated nucleic acid molecule according to claim 1 wherein the fluorophore is a dye, a protein, or a semiconductor nanocrystal.

4. The isolated nucleic acid molecule according to claim 1 wherein one end comprises a C6 thiol-modified base.

5. The isolated nucleic acid molecule according to claim 1 wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

20 or SEQ ID NO;

21.

6. The isolated nucleic acid molecule according to claim 5, wherein the isolated nucleic acid molecule consists of SEQ ID NO:

20 or SEQ ID NO;

21.

7. A sensor chip comprising:

a fluorescence quenching surface;

a first nucleic acid molecule according to claim 1 having first and second ends, and being tethered at the first end to the fluorescence quenching surface; and

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

whereby when the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface 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 fluorophore are substantially free of quenching by the fluorescence quenching surface.

8. The sensor chip according to claim 7 further comprising:

a second nucleic acid molecule that is different from the first nucleic acid molecule and specifically binds to nucleic acids from only one species of methicillin-resistant Staphylococcus, has first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and

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

whereby when the second nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the second fluorophore, and when the second nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the second fluorophore are substantially free of quenching by the fluorescence quenching surface.

9. The sensor chip according to claim 8 wherein the first nucleic acid molecule comprises one or both of SEQ ID NO:

20 and SEQ ID NO;

21 and wherein the second nucleic acid molecule comprises;

one or more of the nucleotide sequences of SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, and SEQ ID NO;

27;

one or more of the nucleotide sequences of SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, and SEQ ID NO;

31; and

one or more of the nucleotide sequences of SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34 SEQ ID NO;

35 and SEQ ID NO;

36.

10. The sensor chip according to claim 8 wherein the second nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO:

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

32, SEQ ID NO;

33, or SEQ ID NO;

36.

11. The sensor chip according to claim 8 wherein the second nucleic acid molecule is capable of distinguishing S. aureus from other methicillin-resistant Staphylococcus.

12. The sensor chip according to claim 8 wherein the second nucleic acid molecule is capable of distinguishing S. sciuri from other methicillin-resistant Staphylococcus.

13. The sensor chip according to claim 8 wherein the second nucleic acid molecule is capable of distinguishing S. epidermidis from other methicillin-resistant Staphylococcus.

14. The sensor chip according to claim 8, wherein the first and second nucleic acid molecules are present in the form of an array.

15. A biological sensor device comprising:

a sensor chip according to claim 7;

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

a detector positioned to detect fluorescent emissions by the first fluorophore.

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

exposing the sensor chip according to claim 7 to a sample under conditions effective to allow any target nucleic acid molecule in the sample to hybridize to the first nucleic acid molecule, causing the first nucleic acid molecule into the non-hairpin conformation;

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

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

17. A method of detecting the presence of a methicillin-resistant strain of Staphylococcus in a sample comprising:

providing the sensor chip according to claim 7, wherein the first nucleic acid molecule hybridizes specifically to a target nucleic acid molecule from a methicillin-resistant strain of Staphylococcus;

exposing the sensor to a sample under conditions effective to allow any target nucleic acid molecule in the sample to hybridize to the first nucleic acid molecule, causing the first nucleic acid molecule into the non-hairpin conformation;

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

determining whether or not the sensor chip emits fluorescent emissions of the first fluorophore upon said illuminating, wherein fluorescent emission by the sensor chip indicates that the first nucleic acid molecule is in the non-hairpin conformation and therefore that the target nucleic acid molecule is present in the sample, thereby identifying presence of the methicillin-resistant strain of Staphylococcus in the sample as tested.

18. An isolated nucleic acid molecule that is less than about 60 nucleotides in length, is characterized by being able to self-anneal into a hairpin conformation, and comprises the nucleotide sequence of SEQ ID NO:

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, or SEQ ID NO;

36.

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

exposing the sensor chip according to claim 18 to a sample under conditions effective to allow any target nucleic acid molecule in the sample to hybridize to its corresponding nucleic acid probe molecules, causing the nucleic acid probe molecule to adopt the non-hairpin conformation;

illuminating the sensor chip with light sufficient to cause emission of fluorescence by the fluorophores; and

determining whether or not the sensor chip emits fluorescent emissions of the fluorophores upon said illuminating, wherein fluorescent emission by the fluorophore of one nucleic acid probe molecule indicates that the target nucleic acid molecule for the one probe molecule is present in the sample.

19. A sensor chip comprising:

a fluorescence quenching surface;

two or more nucleic acid probe molecules each having first and second ends, being characterized by the ability to self-anneal into a hairpin conformation, and being tethered at the first end to the fluorescence quenching surface, wherein the two or more nucleic acid molecules comprise different nucleotide sequences selected from the group consisting of SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, and SEQ ID NO;

36; and

a fluorophore bound to the second end of each nucleic molecule;

whereby when nucleic acid probe molecules are in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore, and when the nucleic acid molecules are in a non-hairpin conformation fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface.

20. The sensor chip according to claim 19, wherein the two or more nucleic acid probe molecules are present in the form of an array.

21. A biological sensor device comprising:

a sensor chip according to claim 19;

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

a detector positioned to detect fluorescent emissions by the fluorophore.