Assays
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.
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.