Assays

• US 8,846,313 B2
• Filed: 11/06/2007
• Issued: 09/30/2014
• Est. Priority Date: 11/06/2006
• Status: Active Grant

• Current Assignee: Alere Technologies GmbH (Abbott Laboratories)
• Original Assignee: Clondiag GmbH (Abbott Laboratories)
• Inventors: Steinmetzer, Katrin, Ermantraut, Eugen, Schulz, Torsten, Kaiser, Thomas, Ullrich, Thomas
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US12/513,597
• Publication Number:
  US 20110014606A1
• Time in Patent Office: 2,520 Days
• Field of Search: 435/6.1, 435/6.12, 435/91.2, 435/287.2, 436/164, 536/24.3, 536/24.33
• US Class Current: 435/6.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00695 Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• 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: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C12Q 1/6813 : Hybridisation assays
• CPC Class Codes: C12Q 1/6825 : Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/701 : Specific hybridization probes
• CPC Class Codes: C12Q 1/703 : Viruses associated with AIDS
• CPC Class Codes: C12Q 2600/158 : Expression markers

Abstract

A device comprising a rigid substrate, a flexible cover element at least partially covering the substrate, a first structure formed in the substrate, adapted for accommodating liquids and adapted for releasing contents of one or more cells, spores, or viruses, the contents including the target molecules, a second structure formed in the substrate, adapted for accommodating liquids and comprising at least one binding member adapted for capturing the target molecules and for determining a value indicative of the presence and/or amount of the target molecules, a micro fluidic network interconnecting at least the first structure and the second structure, and an actuator member adapted for effecting a fluid flow between the first structure and the second structure by pressing the flexible cover element against the substrate to selectively close a portion of the micro fluidic network.

Claims

1. A method, comprising

(a) providing:

an amount of a reporter compound;

a first binding member being configured to bind an anchor group of a capture molecule;

a second binding member capable of capturing the reporter compound;

an amount of a target nucleic acid capable of forming complexes with the reporter compound, the forming of complexes with the reporter compound inhibiting capturing of the reporter compound by the second binding member; and

an amount of capture molecules wherein each capture molecule comprises a binding portion specific to a region of the target nucleic acid and an anchor group;

(b) forming complexes each comprising a target nucleic acid and a capture molecule;

(c) contacting the complexes with the first binding member to bind the complexes to the first binding member;

(d) releasing at least a subset of the amount of target nucleic acid from the first binding member;

(e) forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid;

(f) capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the second binding member;

(g) determining a value indicative of amount of reporter compound captured on the second binding member; and

(h) determining a value indicative of the amount of target nucleic acid based on the value indicative of the amount of reporter compound captured on the second binding member.

2. The method of claim 1, further comprising:

releasing the remaining subset of the amount of reporter compound from the second binding member after the step of determining a value indicative of the amount of reporter compound captured on the second binding member;

forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid;

capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the second binding member; and

determining the value indicative of the amount of reporter compound captured on the second binding member.

3. The method of claim 2, further comprising performing the steps of releasing, forming complexes, capturing, and determining a number N additional times, where N is an integer greater than or equal to 1, 5, 10 or 20.

4. The method of claim 2, wherein the reporter specific capture molecules are oligonucleotides.

5. The method of claim 4, wherein the different reporter specific capture molecules are arranged on different locations with respect to the second binding member.

6. The method of claim 5, wherein reporter compounds are captured on the second binding member by forming complexes with the reporter specific capture molecules.

7. The method of claim 6, wherein at least a part of an interaction site of the reporter compound being capable of forming a complex with a target nucleic acid is also capable of forming a complex with a reporter specific capture molecule.

8. The method of claim 7, wherein the reporter specific capture molecules and the target nucleic acid compete for forming a complex with the reporter compound.

9. The method of claim 1, further comprising subjecting the target nucleic acid to amplification.

10. The method of claim 9, wherein amplification of the target nucleic acid is initiated prior to the step of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid.

11. The method of claim 1, wherein the value indicative of the amount of reporter compound captured on the second binding member is determined before the forming of complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid and the capturing of a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the second binding member are in chemical equilibrium.

12. The method of claim 11, wherein the value indicative of the amount of reporter compound captured on the second binding member is determined 1 seconds to 120 seconds after initiating the steps of forming complexes of a subset of the amount of reporter compound with at least a subset of the amount of target nucleic acid and of capturing a remaining subset of the amount of reporter compound not in complex with a target nucleic acid on the second binding member.

13. The method of claim 1, wherein the second binding member comprises one or more different reporter specific capture molecules being capable of capturing a reporter compound on the second binding member.

14. A method for detecting a target nucleic acid in a liquid, comprising:

introducing a liquid into a reaction chamber of a microfluidic device, the liquid comprising a target polynucleotide,

binding the target polynucleotide to a first binding member located within the reaction chamber,

amplifying the target polynucleotide within the reaction chamber to form amplicons, the amplification being performed in the presence of a reporter compound,

binding a subset of the reporter compound to amplicons within the reaction chamber,

binding a remaining subset of the reporter compound to a second binding member within the reaction chamber, and

detecting the remaining subset of the reporter compound bound to the second binding member;

determining a value indicative of the presence and/or amount of reporter compound captured on the second binding member; and

determining a value indicative of the amount of target nucleic acid based on the value indicative of the amount of reporter compound captured on the second binding member.