Apparatus for automated assay of DNA probe and method for assaying nucleic acid in sample

• US 5,538,849 A
• Filed: 12/29/1993
• Issued: 07/23/1996
• Est. Priority Date: 12/29/1992
• Status: Expired due to Term

• Current Assignee: Toyoda Gosei Company Limited (Toyota Motor Corporation)
• Original Assignee: Toyoda Gosei Company Limited (Toyota Motor Corporation)
• Inventors: Nakajima, Takashi, Tsuruoka, Makoto, Kobata, Hiromichi, Chikanari, Ken-ichi, Yoshimoto, Misao, Uematsu, Hiroaki
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/174,308
• Time in Patent Office: 937 Days
• Field of Search: 435/6, 435/291, 422/82.05, 422/55, 422/62, 422/63, 422/93, 422/100, 422/104, 422/173, 422/174, 436/43, 436/48, 436/49, 436/94, 436/805, 436/807, 436/808, 536/23.1, 536/24.3, 935/76, 935/77, 935/85, 935/86, 935/87
• US Class Current: 435/6.19
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: G01N 35/026 having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/114165 with step of insertion or removal from test line
• CPC Class Codes: Y10T 436/114998 with treatment or replacement of aspirator element [e.g., cleaning, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An apparatus is presented for an automated assay of a DNA probe, which is performed by hybridizing a nucleic acid in a sample with a DNA probe marked with an enzyme and optically measuring a change that occurs in the substrate by catalysis of the enzyme which is bound with the DNA probe. The DNA probe hybridizes with the nucleic acid in the sample to assay the nucleic acid in the sample. Comprising (A) a sample-reagent unit, (B) a sample-reagent dispense unit, (C) a reaction vessel transport unit, (D) a hybridization unit, (E) a B/F separation unit, and (F) a light measurement unit; also presented is a method for assaying a nucleic acid in a sample by the apparatus of the present invention. According to the measurement apparatus of the present invention, a continuous and highly precise measurement is attainable. In particular, the light-shielding structure of the light measurement unit exhibits high levels of light-shielding effect and contributes to efficient light measurement and prevention of degradation of light receiving elements.

Claims

1. An apparatus for an automated assay of a sample using a DNA probe, which is performed by hybridizing a nucleic acid in a sample with a DNA probe marked with an enzyme and optically measuring a change that occurs in a substrate by catalysis of said enzyme which is bound with said probe hybridized with said nucleic acid in said sample to assay said nucleic acid in said sample, comprising:

A--a sample-reagent unit, comprising said sample, reagents, and reaction vessel;

B--a sample-reagent dispense unit in communication with said sample-reagent unit, comprising means to access and deliver said sample and reagents into and out of said reaction vessel;

C--a reaction vessel transport unit in communication with said sample-reagent dispense unit, comprising means for transporting said reaction vessel;

D--a hybridization unit in communication with said reaction vessel transport unit, comprising means for shaking and hybridizing said sample with said DNA probe in said reaction vessel;

E--a B/F separation unit composed of said sample-reagent unit A, said sample-reagent dispense unit B, and said hybridization unit D, the combination of which comprises means to separate unhybridized (F) from hybridized (B) sample nucleic acid that is bound to said reaction vessel; and

F--a light measurement unit in communication with said hybridization unit, comprising means to measure the optical change of the contents of said reaction vessel.

2. The apparatus of claim 1, wherein said sample-reagent unit A comprises (1) a sample tube comprising said sample, (2) a reaction vessel in which a capturing DNA probe has been immobilized, (3) a reagent pack comprising said DNA probe marked with said enzyme and a substrate, (4) a reagent cassette composed of a mixing tank and a holder in which said reaction vessel is movably held, (5) an alkaline denaturant reservoir, and (6) a washing solution reservoir.

3. The apparatus of claim 2, wherein said sample-reagent dispense unit B comprises (1) a freely movable arm, wherein said arm moves to the directions of the X-Y-Z axes, (2) means to move said arm, (3) a tip nozzle set in said arm, (4) a tip for said tip nozzle, and (5) a tip rack holding said tip wherein said tip is set at the head portion of said tip nozzle by moving said arm, thereby enabling suction and injection of said sample in said sample tube and said reagent in said reagent pack.

4. The apparatus of claim 3, wherein said reaction vessel transport unit C comprises (1) means for holding a vessel wherein said holding means is formed on said arm, (2) means to move said arm, and (3) means to transport said reaction vessel, wherein said reaction vessel is carried by said holding means to a stock part of a measurement rack.

5. The apparatus of claim 4, wherein said hybridization unit D comprises (1) means to transport said measurement rack from said stock part to an incubator and then back to said stock part, wherein said measurement rack carries reaction vessels, said incubator includes a heating means and a shaking means, and (2) means for sequentially sucking said sample from said sample-reagent dispense unit B and said DNA probe and respectively dispensing same into a reaction vessel located in said measurement rack.

6. The apparatus of claim 5, wherein said B/F separation unit E comprises (1) a discharge nozzle and a washing solution nozzle set in said arm, (2) means for sucking the content of said reaction vessel in said discharge nozzle and for sucking said washing solution from said washing solution reservoir with said washing solution nozzle and injecting said solution into said reaction vessel, and (3) said transport means of said hybridization unit, wherein said content of said reaction vessel is discharged outside said reaction vessel and said washing solution is injected into said reaction vessel.

7. The apparatus of claim 6, wherein said light measurement unit F comprises (1) means for sucking and injecting said substrate from said reagent pack into said reaction vessel after B/F separation, (2) said transport means, and (3) means for measuring an optical change in said reaction vessel.

8. The apparatus of claim 7, wherein said optical measuring means comprises (1) an emitted light detection part having a light receiving part at the head portion thereof, (2) a hollow light-shielding member movable along the peripheral surface of said light receiving part and said reaction vessel comprising (3) a sample and disposed with its opening in opposite relation to said head portion, wherein said hollow light-shielding member moves to cover a gap formed by said head portion and an upper end of said reaction vessel during said optical measurement.

9. The apparatus of claim 7, wherein said optical measuring means comprises (1) an emitted light detection part, (2) a tubular light receiving part to introduce a light emitted by said sample to said emitted light detection part, (3) a hollow light-shielding member movable along the peripheral surface of said tubular light receiving part, (4) a movable shutter that opens a light path through said tubular light receiving party only when an emitted light measurement is underway, and (5) said reaction vessel further comprising a sample and disposed with its opening in opposite relation to said tubular light receiving part.

10. The apparatus of claim 9, wherein said emitted light detection part comprises (1) a means for photoelectric transfer of said emitted light and (2) a means for determining an emission amount from said photoelectric transfer value.

11. A method for assaying a nucleic acid in a sample by the apparatus of claim 1, comprising the following four steps:

1. a sample denaturing step wherein,

(a) a sample in a sample tube and an alkaline denaturant from an alkaline denaturant reservoir are sequentially sucked with a tip nozzle equipped with a tip of the sample-reagent dispense unit, simultaneously dispensed to a mixing tank in a reagent cassette and mixed to make the nucleic acid in the sample single-stranded;

2. a hybridization step wherein,

(a) a reaction vessel, in which a capturing probe has been immobilized, is transported from a reagent cassette into a holder in a measurement rack in a stock part of a hybridization unit by means of a reaction vessel transport unit, and

(b) the single-stranded nucleic acid of step 1(a) and a DNA probe marked with an enzyme, which was sucked from the reagent pack in the reagent cassette with a tip nozzle equipped with a tip, are dispensed to said reaction vessel, and the measurement rack is passed to an incubator part by a circulation mechanism, wherein said incubator part includes heating and shaking means to allow hybridization in said reaction vessel;

13. A method for determining a nucleic acid in a sample by the apparatus of claim 1, comprising the following four steps:

1. a sample denaturing step wherein,

(a) a sample in a sample tube and an alkaline denaturant from an alkaline denaturant reservoir are sequentially sucked with a tip nozzle equipped with a tip of a sample-reagent dispense unit, and simultaneously dispensed to a mixing tank in a reagent cassette and mixed to make the nucleic acid in the sample single-stranded;

2. a hybridization step wherein,

(a) a reaction vessel, in which a capturing probe has been immobilized, is transported from a reagent cassette into a holder in a measurement rack in a stock part of a hybridization unit by means of a reaction vessel transport unit,

(b) the single-stranded nucleic acid of step 1(a) is dispensed to said reaction vessel and the measurement rack is passed to the incubator part by a circulation mechanism, wherein said incubator part includes heating and shaking means to allow hybridization in the reaction vessel, and

(c) a DNA probe marked with an enzyme, which was sucked from the reagent pack in the reagent cassette with a tip nozzle equipped with a tip of the dispense unit, is injected into said reaction vessel, and the measurement rack is passed through an incubator part by a circulation mechanism, wherein hybridization is allowed in said reaction vessel;

3. a B/F separation and washing step wherein,

(a) the content of the reaction vessel after the hybridization is sucked with a discharge nozzle of the sample-reagent dispense unit and discharged, a washing solution is sucked from a washing solution reservoir with a washing solution nozzle of said dispense unit and injected into said reaction vessel, the measurement rack is passed to an incubator part by a circulation mechanism, wherein said measurement rack is heated and shaken to allow B/F separation in said reaction vessel, and

(b) the washing solution is sucked for discharge from the reaction vessel after the B/F separation with the discharge nozzle; and

4. a light measurement step wherein,

(a) an enzyme substrate is sucked from a reagent pack in the reagent cassette with a tip nozzle equipped with a tip of the sample-reagent dispense unit and injected into the reaction vessel, the measurement rack is passed to an incubator part by a circulation mechanism of the hybridization, wherein said measurement rack is heated, and

(b) the measurement rack is transported to a light measurement part by the circulation device, wherein said light measurement part measures light change in the reaction vessel by means of a light measurement device.

12. a B/F separation and washing step wherein,

(a) the content of the reaction vessel after the hybridization is sucked with a discharge nozzle of the sample-reagent dispense unit and discharged, a washing solution is sucked from a washing solution reservoir with a washing solution nozzle of said dispense unit and injected into said reaction vessel, the measurement rack is passed to an incubator part by a circulation mechanism, wherein said measurement rack is heated and shaken to allow B/F separation in said reaction vessel, and

(b) the washing solution is sucked with the discharge nozzle from the reaction vessel after the B/F separation and discharged;

and

4. a light measurement step wherein,

(a) an enzyme substrate is sucked from a reagent pack in the reagent cassette with a tip nozzle equipped with a tip of the sample-reagent dispense unit and injected into the reaction vessel, the measurement rack is passed to an incubator part by a circulation mechanism, wherein said measurement rack is heated, and

(b) the measurement rack is transported to a light measurement part by the circulation mechanism, wherein said light measurement part measures light change in the reaction vessel by means of a light measurement device.