Nucleic acid concentration quantitative analysis chip, nucleic acid concentration quantitative analysis apparatus, and nucleic acid concentration quantitative analysis method

• US 20050164286A1
• Filed: 03/18/2005
• Published: 07/28/2005
• Est. Priority Date: 02/26/2003
• Status: Abandoned Application

• Current Assignee: Kabushiki Kaisha Toshiba (Toshiba Corporation)
• Original Assignee: Kabushiki Kaisha Toshiba (Toshiba Corporation)
• Inventors: Hashimoto, Koji, Funaki, Hideyuki, Okada, Jun, Gemma, Nobuhiro, Hongo, Sadato, O'uchi, Shin-ichi, Takahashi, Masayoshi
• Application Number:
  US11/082,877
• Publication Number:
  US 20050164286A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/143 Quality control, feedback systems
• CPC Class Codes: B01L 2200/148 Specific details about calibrations
• CPC Class Codes: B01L 2300/024 Storing results with means integrated into the container
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The present invention includes a plurality of working electrodes on which the same type of nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized and which have different sensor areas and a normalization circuit which normalizes detection signals obtained by the working electrodes with respect to the respective sensor areas.

Claims

1. A nucleic acid concentration quantitative analysis chip comprising:

a plurality of nucleic acid sensors having different sensor areas on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized; and

a first normalization unit which normalizes first detection signals obtained by the nucleic acid sensors with respect to the respective sensor areas.

2. The nucleic acid concentration quantitative analysis chip according to claim 1, further comprising:

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a second normalization unit which normalizes second detection signals obtained by the background level sensors with respect to the respective sensor areas; and

a subtraction circuit which subtracts a second output signal of the second normalization unit from a first output of the first normalization unit.

3. The nucleic acid concentration quantitative analysis chip according to claim 1, further comprising:

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a second normalization circuit which normalizes second detection signals obtained by the background level sensors with respect to the respective sensor areas;

a subtraction unit which subtracts a second output signal of the second normalization unit from a first output of the first normalization unit; and

a current-to-voltage conversion unit which converts an output signal current of the subtraction unit to a voltage.

4. The nucleic acid concentration quantitative analysis chip according to claim 1, wherein the first normalization unit comprises:

a first current mirror which duplicates and amplifies a first current of the first detection signal detected from the nucleic acid sensor and outputs that amplified current if the first current value is positive; and

a second current mirror which duplicates and amplifies the first current and outputs that amplified current if the first current value is negative.

5. The nucleic acid concentration quantitative analysis chip according to claim 1, further comprising:

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a second normalization circuit which normalizes second detection signals obtained by the background level sensors with respect to the respective sensor areas;

a subtraction unit which subtracts an second output signal of the second normalization unit from an first output of the first normalization unit,

wherein the first normalization circuit comprises;

a first current mirror which duplicates and amplifies a first current of the first detection signals detected from the nucleic acid sensor if the first current value is positive; and

a second current mirror which duplicates and amplifies the first current if the first current value is negative,

the second normalization circuit comprises;

a third current mirror which duplicates and amplifies a second current of the second detection signals detected by the background level sensor if the second current value is positive; and

a fourth current mirror which duplicates and amplifies the second current if the second current value is negative, and

the subtraction circuit subtracts a third output current of the third current mirror from a first output current of the first current mirror and subtracts a fourth output current of the fourth current mirror from a second output current of the second current mirror.

6. The nucleic acid concentration quantitative analysis chip according to claim 1, further comprising:

a plurality of cells, each of which house one or more of the nucleic acid sensors, which are separated from one another in accordance with the areas of the nucleic acid sensors.

7. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor, and a first measurement range of the first sensor overlaps with a second measurement range of the second sensor.

8. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor, a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and if a dynamic range d₁(dec) of the first sensor is

defined as a first ratio of a first upper end to a first lower end of the first the measurement range, the number of first nucleic acid probes of the first sensor is N₁, the number of second nucleic acid probes of the second sensor is N₂, a second ratio of a second upper end to a second lower end of a nucleic acid concentration region in which the first the measurement range overlaps with the second measurement range is d_1,2(dec), and a ratio d_1,2/d₁ of d_1,2 to d₁ is γ

(0≦

γ

<

1), the following is satisfied;

${10}_1^d<br><br>\left(1-\mathrm{\gamma <br><br>}\right)=\frac{N_1}{N_2}.$

9. The nucleic acid concentration quantitative analysis chip according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor, a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and

if a dynamic range d₁(dec) of the first sensor is defined as a first ratio of a first upper end to a first lower end of the first the measurement range, the number of first nucleic acid probes of the first sensor is N₁, the number of second nucleic acid probes of the second sensor is N₂, a second ratio of a second upper end to a second lower end of a nucleic acid concentration region in which the first the measurement range overlaps with the second measurement range is d_1,2(dec), a ratio d_1,2/d₁ of d_1,2 to d₁ is γ

(0≦

γ

<

1), the following is satisfied;

${10}_1^d<br><br>\left(1-\mathrm{\gamma <br><br>}\right)=\frac{N_1}{N_2},$

and the value γ

satisfies γ

≦

0.85.

10. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor, a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and

if a dynamic range d₁(dec) of the first sensor is defined as a first ratio of a first upper end to a first lower end of the first the measurement range, the number of first nucleic acid probes of the first sensor is N₁, the number of second nucleic acid probes of the second sensor is N₂, a second ratio of a second upper end to a second lower end of a nucleic acid concentration region in which the first the measurement range overlaps with the second measurement range is d_1,2(dec), a ratio d_1,2/d₁ of d_1,2 to d₁ is γ

(0 ≦

γ

<

1), the following is satisfied;

${10}_1^d<br><br>\left(1-\mathrm{\gamma <br><br>}\right)=\frac{N_1}{N_2},$

and the first and second dynamic ranges d₁ and d₂ satisfy a relation of d₁=d₂.

11. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor,

a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and

if first and second electrode areas of the first and second sensors are defined as S₁ and S₂ respectively, a relation of 0.05≦

S₂/S₁≦

0.5 is satisfied in a case where a number of the nucleic acid probe molecules proportional to the electrode area are immobilized on the first and second sensors respectively.

12. The nucleic acid concentration quantitative analysis chip according to claim 1, wherein the nucleic acid sensor comprises a first sensor, a second sensor having an electrode area smaller than that of the first sensor, and a third sensor having an electrode area smaller than that of the second sensor, a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and

if the electrode areas of the first to third sensors are defined as S₁, S₂, and S₃, a relation of S₂/S₁=S₃/S₂ is satisfied in a case where the nucleic acid probes are immobilized on the first to third sensors in proportion to the electrode areas.

13. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor and a second sensor having an electrode area smaller than that of the first sensor,

a first measurement range of the first sensor overlaps with a second measurement range of the second sensor,

if first and second electrode areas of the first and second sensors are defined as S₁ and S₂, a relation of 0.05≦

S₂/S₁≦

0.5 is satisfied in a case where a number of nucleic acid probe molecules proportional to the electrode area are immobilized on the first and second sensors, and

sensor areas of the plurality of nucleic acid sensors substantially form a geometric progression.

14. The nucleic acid concentration quantitative analysis apparatus according to claim 1, wherein the nucleic acid sensor comprises a first sensor, a second sensor having an electrode area smaller than that of the first sensor, and a third sensor having an electrode area smaller than that of the second sensor, a first measurement range of the first sensor overlaps with a second measurement range of the second sensor, and

if the electrode areas of the first to third sensors are defined as S₁, S₂, and S₃, a relation of 0.05≦

S₂/S₁=S₃/S₂≦

0.5 is satisfied in a case where the nucleic acid probes are immobilized on the first to third sensors in proportion to the electrode areas.

15. A nucleic acid concentration quantitative analysis apparatus comprising:

a plurality of nucleic acid sensors having different sensor areas on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized;

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a first normalization unit which normalizes first detection signals obtained by the nucleic acid sensors with respect to the respective sensor areas;

a second normalization unit which normalizes second detection signals obtained by the background level sensors with respect to the respective sensor areas;

a first current-to-voltage conversion unit which converts a first output signal current of the first normalization unit to a first output voltage;

a second current-to-voltage conversion unit which converts a second output signal current of the second normalization unit to a second output voltage;

an A/D conversion unit which A/D converts the first output voltage to generate first digital data and which A/D converts the second output voltage to generate second digital data; and

a subtraction unit which subtracts the second digital data from the first digital data.

16. A nucleic acid concentration quantitative analysis apparatus comprising:

a plurality of nucleic acid sensors having different sensor areas on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized;

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a first normalization unit which normalizes first detection signals obtained by the nucleic acid sensors with respect to the respective sensor areas;

a second normalization unit which normalizes second detection signals obtained by the background level sensors with respect to the respective sensor areas;

a first current-to-voltage conversion unit which converts a first output signal current of the first normalization unit to a first output voltage;

a second current-to-voltage conversion unit which converts a second output signal current of the second normalization unit to a second output voltage;

a subtraction unit which subtracts the second output voltage from the first output voltage; and

an A/D conversion unit which executes A/D conversion of a third output voltage of the subtraction unit.

17. A nucleic acid concentration quantitative analysis chip comprising:

a plurality of nucleic acid sensors having different sensor areas on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized;

a plurality of background level sensors which have the different sensor areas on which the nucleic acid probes having the nucleic acids complementary to the target nucleic acids are not immobilized;

a subtraction unit which subtracts a second detection signal of the background level sensor from a first detection signal of the nucleic acid sensor; and

a normalization unit which normalizes a subtraction output signal of the subtraction unit.

18. The nucleic acid concentration quantitative analysis chip according to claim 17, further comprising a current-to-voltage conversion unit which converts an output signal current of the normalization unit to a voltage.

19. A nucleic acid concentration quantitative analysis apparatus comprising:

a nucleic acid concentration quantitative analysis chip including;

a plurality of nucleic acid sensors having different sensor areas on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized; and

a first normalization unit which normalizes a detection signal of the nucleic acid sensor with respect to the sensor area to output a normalized signal, and

a nucleic acid concentration calculation device which calculates a nucleic acid concentration based on the normalized signal.

20. The nucleic acid concentration quantitative analysis apparatus according to claim 19, wherein the nucleic acid concentration calculation device compares the normalized signal with a predetermined threshold value to acquire binary bit data with respect to each sensor area, and collates the binary bit data with a judgment table in which binary judgment bit data is associated with a concentration of the nucleic acid with respect to each sensor area beforehand to determine the nucleic acid concentration.

21. The nucleic acid concentration quantitative analysis apparatus according to claim 19, further comprising saturated level sensors having different sensor areas on which double stranded nucleic acids composed of the target nucleic acid and the probe nucleic acid are immobilized.

22. A nucleic acid concentration quantitative analysis method comprising:

normalizing detection signals of a plurality of nucleic acid sensors on which nucleic acid probes each having a nucleic acid complementary to a target nucleic acid are immobilized and which have different sensor areas with respect to sensor areas to output a normalized signal; and

calculating a nucleic acid concentration based on the normalized signal.