Deoxyribonucleic acid measuring apparatus and method of measuring deoxyribonucleic acid

• US 20070059741A1
• Filed: 07/24/2006
• Published: 03/15/2007
• Est. Priority Date: 09/15/2005
• Status: Active Grant

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Ishige, Yu, Kamahori, Masao
• Granted Patent: US 7,838,226 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

With an insulated gate field effect transistor in which deoxyribonucleic acid (DNA) probes are immobilized on a gold electrode, extension reaction on the gold electrode is performed with DNA polymerase to directly measure an increased amount of a phosphate group caused by the extension reaction, that is, negative charge, by means of a current change between a source and a drain of the insulated gate field effect transistor. Thus, presence/absence of hybridization of target DNAs with the DNA probes, and presence/absence of the extension reaction are detected. Optimum immobilization density of the DNA probes on the gold electrode is set at 4×10¹² molecules/cm². To reduce surface potential fluctuation caused by external variation (influences of foreign substances), which is a problem when using the gold electrode in a solution, a high-frequency voltage equal to or above 1 kHz is applied between the gold electrode and a reference electrode by a power source.

Claims

1. A deoxyribonucleic acid (DNA) measuring apparatus comprising:

a field effect transistor including a gold electrode contacting with a measurement solution containing DNA polymerase, DNA probes being immobilized on a surface of the gold electrode;

a reference electrode contacting with the measurement solution;

means which applies a high-frequency voltage between the gold electrode and the reference electrode;

means which injects sample DNAs into the measurement solution; and

means which injects at least one type of deoxyribonucleoside triphosphate selected from a group consisting of dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate), or a deoxyribonucleoside triphosphate derivative.

2. The DNA measuring apparatus according to claim 1, wherein the gold electrode is connected to a gate of the field effect transistor with a conductive wire.

3. The DNA measuring apparatus according to claim 1, wherein a source portion, a drain portion, and a channel portion of the field effect transistor are covered with a light-shielding member.

4. The DNA measuring apparatus according to claim 3, wherein the light-shielding member is a conductive member.

5. The DNA measuring apparatus according to claim 3, wherein the light-shielding member is grounded.

6. The DNA measuring apparatus according to claim 3, wherein the light-shielding member is made of any of aluminum and gold.

7. The DNA measuring apparatus according to claim 1, wherein the high-frequency voltage has a frequency not lower than 1 kHz.

8. The DNA measuring apparatus according to claim 1, wherein at least two sets each consisting of the gold electrode and any of the reference electrode and a pseudo reference electrode are formed on one plane to face each other.

9. The DNA measuring apparatus according to claim 1, wherein the DNA probes are immobilized on the surface of the gold electrode with alkanethiol binding to one end of each of the DNA probes.

10. The DNA measuring apparatus according to claim 1, wherein the deoxyribonucleoside triphosphate derivative is a derivative including an additional portion carrying negative charge which is bound to a side chain of a constituent base.

11. The DNA measuring apparatus according to claim 10, wherein a part of the additional portion contains a phosphate group.

12. The DNA measuring apparatus according to claim 1, wherein the measurement solution contains a deoxyribonucleoside triphosphate decomposing enzyme.

13. The DNA measuring apparatus according to claim 1, wherein the DNA probes contains a base sequence of a universal primer used for sequencing.

14. A method of measuring DNAs comprising the steps of:

contacting a gold electrode of a field effect transistor with a measurement solution containing DNA polymerase, DNA probes being immobilized on a surface of the gold electrode;

applying a high-frequency voltage between the gold electrode and a reference electrode contacting with the measurement solution;

injecting sample DNAs into the measurement solution;

injecting at least one type of a deoxyribonucleoside triphosphate selected from a group consisting of dATP, dCTP, dGTP, and dTTP, or a deoxyribonucleoside triphosphate derivatives after a certain period of time; and

measuring an electric characteristic of the transistor changing before and after binding the sample DNAs to the DNA probes, or before and after extension of the DNA probes; and

detecting any of presence/absence of the sample DNAs bound to the DNA probes, and a base sequence.

15. The method of measuring DNAs according to claim 14, wherein

the measurement solution containing the DNA polymerase further includes a deoxyribonucleoside triphosphate decomposing enzyme;

at least one type of the deoxyribonucleoside triphosphate selected from a group consisting of dATP, dCTP, dGTP, and dTTP, or a deoxyribonucleoside triphosphate derivatives is injected repeatedly; and

the base sequence of each of the sample DNAs is detected by measuring the electric characteristic of the field effect transistor changing before and after extension of the DNA probes.

16. The method of measuring DNAs according to claim 14, wherein the gold electrode is connected to a gate of the field effect transistor with a conductive wire.

17. The method of measuring DNAs according to claim 14, wherein the high-frequency voltage has a frequency equal to or above 1 kHz.

18. The method of measuring DNAs according to claim 14, wherein the DNA probes are immobilized on the surface of the gold electrode with alkanethiol binding to one end of each of the DNA probes.

19. The method of measuring DNAs according to claim 14, wherein the deoxyribonucleoside triphosphate derivative is a derivative including an additional portion carrying negative charge which is bound to a side chain of a constituent base.

20. The method of measuring DNAs according to claim 19, wherein a part of the additional portion includes a phosphate group.

21. The method of measuring DNAs according to claim 14, wherein the DNA probes have a base sequence of a universal primer used for sequencing.

22. The method of measuring DNAs according to claim 14, wherein any of a voltage between a source and a drain, and a gate voltage change is measured before and after binding the sample DNAs to the DNA probes or before and after extension of the DNA probes while maintaining a drain current at a constant level.