Device and method for multiple analyte detection

• US 7,833,711 B2
• Filed: 04/07/2006
• Issued: 11/16/2010
• Est. Priority Date: 04/03/1996
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Kowallis, Reid B., Albin, Michael, Winn-Deen, Emily S., Ragusa, Robert P., Raysberg, Yefim, Woudenberg, Timothy M.
• Primary Examiner(s): Chin; Christopher L
• Application Number:
  US11/400,611
• Publication Number:
  US 20060183151A1
• Time in Patent Office: 1,684 Days
• Field of Search: 435/4, 435/6, 436/501, 436/518, 436/164, 436/166, 436/807, 422/50, 422/61, 422/68.1
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0642 Filling fluids into wells by specific techniques
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50851 specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/08 using multifield media
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/808 : Automated or kit
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more of a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.

Claims

1. A method for detecting fluorescence in a liquid sample, comprising:

introducing a liquid sample comprising a target nucleic acid sequence into a chamber loaded with detection reagents prior to introducing the liquid sample, the detection reagents comprising a first unlabeled oligonucleotide;

a second unlabeled oligonucleotide; and

a fluorescently-labeled oligonucleotide;

causing a first reaction in the chamber, between the target nucleic acid sequence and both the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, under conditions effective to selectively bind each of the first unlabeled oligonucleotide and the second unlabeled oligonucleotide to the target nucleic acid sequence and to form a reaction product;

causing a second reaction in the chamber, to occur at the same time the first reaction is occurring, wherein the second reaction comprises a reaction between the fluorescently-labeled oligonucleotide and the reaction product and wherein a second reaction product is formed that emits a detectable fluorescent signal upon excitation thereof;

exciting the second reaction product in the chamber to cause the second reaction product to emit a detectable fluorescent signal from the chamber; and

detecting the detectable fluorescent signal emitted from the chamber.

2. The method of claim 1, wherein the target nucleic acid sequence comprises a single-stranded nucleic acid sequence.

3. The method of claim 1, wherein the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, each bind to a different region of the target nucleic acid sequence.

4. The method of claim 1, wherein the chamber comprises non-immobilized detection reagents.

5. The method of claim 1, wherein the first unlabeled oligonucleotide, the second unlabeled oligonucleotide, and the fluorescently-labeled oligonucleotide are non-immobilized.

6. The method of claim 1, wherein the reacting comprises a polymerase chain reaction.

7. The method of claim 1, wherein the chamber comprises a plurality of chambers.

8. The method of claim 1, wherein the chamber comprises a plurality of chambers connected to a channel.

9. The method of claim 1, wherein introducing a liquid sample comprises subjecting the liquid sample to a vacuum.

10. The method of claim 1, wherein the first unlabeled oligonucleotide and the second unlabeled oligonucleotide comprise a primer pair for polymerase chain reaction amplification.

11. The method of claim 1, wherein the fluorescently-labeled oligonucleotide is a sequence-selective oligonucleotide.

12. The method of claim 11, wherein the sequence-selective oligonucleotide comprises a fluorescer-quencher pair.

13. The method of claim 1, wherein the first oligonucleotide and second oligonucleotide bind selectively to adjacent, contiguous regions of a target nucleic acid.

14. A method for detecting fluorescence in a liquid sample, comprising:

introducing a liquid sample comprising a target nucleic acid sequence into a chamber loaded with detection reagents prior to introducing the liquid sample, the detection reagents comprising a first unlabeled oligonucleotide;

a second unlabeled oligonucleotide; and

a fluorescently-labeled oligonucleotide, wherein introducing the liquid sample into a chamber comprises introducing the liquid sample into a sample-distribution network comprising (i) a sample inlet, (ii) two or more detection chambers, and (iii) a channel in fluid communication with each of the detection chambers and the sample inlet;

causing a first reaction in the chamber, between the target nucleic acid sequence and both the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, under conditions effective to selectively bind each of the first unlabeled oligonucleotide and the second unlabeled oligonucleotide to the target nucleic acid sequence and to form a reaction product;

causing a second reaction in the chamber, to occur at the same time the first reaction is occurring, wherein the second reaction comprises a reaction between the fluorescently-labeled oligonucleotide and the reaction product and wherein a second reaction product is formed that emits a detectable fluorescent signal upon excitation thereof;

exciting the second reaction product in the chamber to cause the second reaction product to emit a detectable fluorescent signal from the chamber; and

detecting the detectable fluorescent signal emitted from the chamber.

15. The method of claim 14, wherein the channel comprises a single channel to which the two or more detection chambers are connected by the fluid connections.

16. A method for detecting fluorescence in a liquid sample, comprising:

introducing a liquid sample comprising a target nucleic acid sequence into a chamber loaded with detection reagents prior to introducing the liquid sample, the detection reagents comprising a first unlabeled oligonucleotide;

a second unlabeled oligonucleotide; and

a fluorescently-labeled oligonucleotide, wherein introducing the liquid sample into a chamber comprises introducing the liquid sample into a sample-distribution network comprising (i) a sample inlet, (ii) two or more detection chambers, and (iii) a channel providing respective dead-end fluid connections between each of the chambers and the sample inlet;

causing a first reaction in the chamber, between the target nucleic acid sequence and both the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, under conditions effective to selectively bind each of the first unlabeled oligonucleotide and the second unlabeled oligonucleotide to the target nucleic acid sequence and to form a reaction product;

causing a second reaction in the chamber, to occur at the same time the first reaction is occurring, wherein the second reaction comprises a reaction between the fluorescently-labeled oligonucleotide and the reaction product and wherein a second reaction product is formed that emits a detectable fluorescent signal upon excitation thereof;

exciting the second reaction product in the chamber to cause the second reaction product to emit a detectable fluorescent signal from the chamber; and

detecting the detectable fluorescent signal emitted from the chamber.

17. The method of claim 16, wherein the channel comprises a single channel to which the two or more detection chambers are connected by the fluid connections.

18. The method of claim 16, wherein the target nucleic acid sequence comprises a single-stranded nucleic acid sequence.

19. The method of claim 16, wherein the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, each bind to a different region of the target nucleic acid sequence.

20. The method of claim 16, wherein the chamber comprises non-immobilized detection reagents.