Methods of concentrating an analyte
View Patent ↗A receptacle having a plurality of interconnected chambers arranged to permit multiple process steps or processes to be performed independently or simultaneously. The receptacles are manufactured to separate liquid from dried reagents and to maintain the stability of the dried reagents. An immiscible liquid, such as an oil, is included to control loading of process materials, facilitate mixing and reconstitution of dried reagents, limit evaporation, control heating of reaction materials, concentrate solid support materials to prevent clogging of fluid connections, provide minimum volumes for fluid transfers, and to prevent process materials from sticking to chamber surfaces. The receptacles can be adapted for use in systems having a processing instrument that includes an actuator system for selectively moving fluid substances between chambers and a detector. The actuator system can be arranged to concentrate an analyte present in a sample. The detector can be used to detect an optical signal emitted by the contents of the receptacle.
1. A method of concentrating an analyte present in a fluid sample provided to a receptacle having a plurality of interconnected chambers, the method comprising the steps of:
(a) forming a first volume in a first chamber of the receptacle, the first volume comprising the fluid sample and a liquid reagent containing a solid support;
(b) immobilizing the analyte on the solid support;
(c) moving an aliquot of the first volume from the first chamber to a directly connected second chamber of the receptacle by applying one or more compressive forces to a flexible portion of the first chamber with one or more actuators, wherein the volume capacity of the second chamber is less than the first volume;
(d) isolating the analyte immobilized on the solid support in the second chamber;
(e) moving non-analyte components of the sample from the second chamber to a directly connected waste chamber of the receptacle by applying a compressive force to a flexible portion of the second chamber with an actuator;
(f) repeating steps (c) through (e) one or more times while retaining immobilized analyte in the second chamber, thereby accumulating concentrated analyte in the second chamber; and
(g) after step (f), moving the concentrated analyte accumulated in the second chamber to a directly connected third chamber by applying a compressive force to a flexible portion of the second chamber with an actuator.
2. The method of claim 1 , wherein steps (c) through (e) are repeated multiple times.
3. The method of claim 1 , wherein the first chamber is a sample receiving chamber having a sample addition port.
4. The method of claim 1 , wherein the liquid reagent is moved to the first chamber from a fourth chamber during step (a).
5. The method of claim 1 , wherein the first volume further comprises an immiscible liquid.
6. The method of claim 5 , wherein the immiscible liquid is an oil.
7. The method of claim 6 , wherein the oil is a mineral oil.
8. The method of claim 5 , wherein the ratio of the immiscible liquid to the liquid reagent is from about 1:10 to about 10:1.
9. The method of claim 5 , wherein the ratio of the immiscible liquid to the liquid reagent is from about 1:3 to about 10:1.
10. The method of claim 1 , wherein the receptacle is comprised of opposed members configured to define the plurality of interconnected chambers.
11. The method of claim 10 , wherein at least one of the opposed members comprises a flexible sheet, the flexible sheet comprising the flexible portion of the first chamber.
12. The method of claim 11 , wherein each of the opposed members comprises a flexible sheet.
13. The method of claim 10 further comprising the step of altering an openable connection directly connecting the first and second chambers from a closed state to an open state when the one or more compressive forces are applied to the flexible portion of the first chamber or when a compressive force is removed from the connection.
14. The method of claim 13 , wherein the plurality of interconnected chambers are defined by seals formed between the opposed members.
15. The method of claim 14 , wherein the first and second chambers are adjacent to each other, and wherein the openable connection is an opening blocked by a burstable seal that is altered from the closed state to the open state when the one or more compressive forces are applied to the flexible portion of the first chamber.
16. The method of claim 15 , wherein the burstable seal is a heat seal.
17. The method of claim 1 , wherein the one or more actuators have compression pads that are arranged to generally conform to the shape of the first chamber.
18. The method of claim 1 , wherein the actuators are pneumatic actuators.
19. The method of claim 13 , wherein the openable connection is altered from the closed state to the open state when a compressive force is removed from the connection.
20. The method of claim 1 , wherein the solid support is a particle or bead.
21. The method of claim 20 further comprising the step of subjecting the solid support to a magnetic field during step (d), the solid support being magnetically-responsive.
22. The method of claim 1 , wherein the analyte remains immobilized on the solid support during step (g).
23. The method of claim 1 , wherein the analyte is a target nucleic acid.
24. The method of claim 23 further comprising, in the third chamber, the step of subjecting the analyte to an amplification reagent and amplification conditions.
25. The method of claim 24 further comprising the step of detecting an amplification product in the third chamber.
26. The method of claim 1 , wherein each of the plurality of interconnected chambers is positioned adjacent to at least one other chamber of the receptacle.
27. The method of claim 1 , wherein step (c) comprises applying a compressive force to a first flexible portion of the first chamber with a first actuator to partition the first chamber into at least two sub-chambers, each sub-chamber containing a portion of the first volume, and applying a compressive force to a flexible portion of one of the sub-chambers with a second actuator to force the portion of the first volume contained in the first sub-chamber into the second chamber.