Methods and apparatuses for gene purification and imaging
View Patent ↗The present disclosure is directed to systems, devices and methods for nucleic acid or protein purification and imaging. A system is provided including a cartridge comprising a sample input area configured to hold a sample, comprising a plurality of hybridized complexes comprising a plurality of target molecules each hybridized with probes and a plurality of non-hybridized probes. The cartridge may also include a first binding chamber configured with first magnetic beads to receive and bind the sample, a first elution channel configured to receive the first magnetic beads and elute the sample from the first magnetic beads, a second binding chamber configured with second magnetic beads to receive and bind the sample, a second elution channel configured to receive the second magnetic beads and elute the sample from the second magnetic beads, and a binding area configured to receive the eluted sample and hold molecules for imaging.
1. A cartridge configured for purifying a hybridized target molecule sample and imaging a hybridized target molecule, comprising:
a sample input area configured to hold a target molecule sample, the sample comprising:
a plurality of hybridized complexes comprising a plurality of target molecules each hybridized with a first probe and a second probe,
a plurality of non-hybridized first probes, and
a plurality of non-hybridized second probes;
a first binding chamber configured to receive or contain a first affinity matrix and to receive the sample, wherein:
the first affinity matrix is functionalized with first molecules configured to bind with the non-hybridized first probes and hybridized complexes of the sample during a first period of time;
the first binding chamber is additionally configured to receive a first buffer to remove non-hybridized second probes from the sample after the non-hybridized first probes and hybridized complexes of the sample bind with the first affinity matrix;
a first elution channel attached to the first binding chamber and a means for heating, and which is configured to receive the first affinity matrix after the first period of time and configured for heating the first affinity matrix to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized first probes;
a second binding chamber configured to receive or contain a second affinity matrix and to receive the first eluted sample, wherein:
the second affinity matrix is functionalized with second molecules configured to bind with the hybridized complexes during a second period of time;
the second binding chamber is additionally configured to receive a second buffer to remove at least non-hybridized first probes;
a second elution channel attached to the second binding chamber and a means for heating, and which is configured to receive the second affinity matrix after the second period of time and configured for heating the second affinity matrix to elute a second eluted sample comprising the plurality of hybridized complexes; and
a binding area having an active binding surface configured to receive the second eluted sample and bind with the hybridized complexes.
2. The cartridge of claim 1 , wherein the target molecule is a nucleic acid or a protein.
3. The cartridge of claim 1 , wherein the first affinity matrix and the second affinity matrix correspond to a first set of magnetic beads and a second set of magnetic beads, respectively.
4. The cartridge of claim 1 , wherein the active binding surface comprises one of streptavidin, an avidin, and oligonucleotides.
5. The cartridge of claim 3 , wherein the cartridge further comprises:
a plurality of buffer input areas,
a plurality of first binding chambers,
a plurality of waste output areas, and
a plurality of bead pads.
6. The cartridge of claim 1 , wherein the first probes include capture probes.
7. The cartridge of claim 1 , wherein the second probes include reporter probes.
8. The cartridge of claim 5 , wherein the bubble vent configured to separate the sample input and the first binding chamber and to eliminate air bubbles.
9. The cartridge of claim 3 , wherein the first magnetic beads include oligonucleotide-coupled magnetic beads.
10. The cartridge of claim 9 , wherein the oligonucleotide-coupled magnetic beads include F magnetic beads.
11. The cartridge of claim 3 , wherein the second magnetic beads include oligonucleotide-coupled magnetic beads.
12. The cartridge of claim 11 , wherein the oligonucleotide-coupled magnetic beads include G magnetic beads.
13. The cartridge of claim 1 , the cartridge being operatively coupled to:
a plurality of off-card buffer input valves operatively coupled to a fluidic manifold; and
a plurality of waste valves.
14. The cartridge of claim 1 , the cartridge further comprising:
a plurality of on-card buffer input valves operatively coupled to a fluidic manifold.
15. The cartridge of claim 13 , wherein the plurality of off-card buffer input valves are configured to receive the first buffer and the second buffer from the fluidic manifold and provide the buffer to the cartridge.
16. The cartridge of claim 1 , wherein the flow of the second eluted sample onto the binding area is done in small steps that are re-ordered based on pressure profiles.
17. The cartridge of claim 1 , wherein the binding area is further configured to receive a solution formulated to immobilize the second eluted sample on the active binding surface after stretching with flow.
18. The cartridge of claim 17 , wherein the solution includes G-hooks, anti-fade media, and fiducials.
19. A cartridge configured for purifying a hybridized target molecule sample and imaging the hybridized target molecule, comprising:
a buffer input area configured to hold a target molecule sample, the sample comprising:
a plurality of hybridized complexes including a plurality of target molecules, and each of reporter probes and capture probes,
a plurality of non-hybridized reporter probes, and
a plurality of non-hybridized capture probes;
a bubble vent configured to separate the sample input and the first binding chamber and to eliminate air bubbles;
a first binding chamber configured to receive or contain F magnetic beads and to receive the sample, wherein:
the F magnetic beads are functionalized with first molecules configured to bind with the non-hybridized reporter probes and hybridized complexes of the sample during a first period of time;
the first binding chamber is additionally configured to receive a first buffer to remove non-hybridized reporter probes from the sample after the non-hybridized reporter probes and hybridized complexes of the sample bind with the F magnetic beads;
a first elution channel attached to the first binding chamber and a means for heating, and which is configured to receive the F magnetic beads after the first period of time and configured for heating the F magnetic beads to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized reporter probes;
a second binding chamber configured to receive or contain G magnetic beads and to receive the first eluted sample, wherein:
the G magnetic beads are functionalized with second molecules configured to bind with the hybridized complexes during a second period of time;
the second binding chamber is additionally configured to receive a second buffer remove at least non-hybridized capture probes;
a second elution channel attached to the second binding chamber and a means for heating, and which is configured to receive the G magnetic beads after the second period of time and configured for heating the G magnetic beads to elute a second eluted sample comprising the plurality of hybridized complexes;
a binding area having an active binding surface configured to receive the second eluted sample and bind with the hybridized complexes;
wherein the cartridge is operatively coupled to:
a plurality of off-card buffer input valves operatively coupled to a fluidic manifold, the plurality of off-card buffer input valves being configured to receive the first buffer and the second buffer from the fluidic manifold and provide the first and buffer to the cartridge; and
a plurality of waste valves configured to collect the first and second buffer from the cartridge.
20. A system for imaging a plurality of hybridized complexes comprising;
a cartridge according to claim 1 ;
a cartridge tray operatively coupled to the system and configured to hold the cartridge;
a first heater operatively coupled to the cartridge;
a second heater operatively coupled to the cartridge;
a magnet operatively coupled to the imaging device below the cartridge tray;
a fluidic manifold operatively coupled to the system above the cartridge tray and configured to hold and control the flow of a plurality of buffers;
a plurality of off-card buffer input valves operatively coupled to the fluidic manifold and the cartridge;
a plurality of waste valves operatively coupled to the system above the cartridge tray; and
an imaging reference surface operatively coupled to the imaging device above the cartridge tray.
21. A method for purifying a hybridized target molecule sample and imaging the hybridized target molecule, comprising:
providing the cartridge of claim 1 ;
receiving a hybridized sample, the sample comprising a plurality of hybridized complexes comprising target molecules hybridized with first probes and second probes, a plurality of non-hybridized first probes, and a plurality of non-hybridized second probes;
binding the non-hybridized first probes and hybridized complexes of the sample to a first affinity matrix in a first binding chamber during a first period of time;
flowing a first buffer into the first binding chamber to remove non-hybridized second probes from the sample after the non-hybridized first probes and hybridized complexes of the sample bind with the first affinity matrix;
directing the first affinity matrix into a first elution channel;
heating the first affinity matrix to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized first probes;
binding the hybridized complexes of the first eluted sample to a second affinity matrix in a second binding chamber during a second period of time;
flowing a second buffer into the second binding chamber to remove the non-hybridized first probes from the first eluted sample after the hybridized complexes bind with the second affinity matrix;
heating the second affinity matrix to elute a second eluted sample comprising the plurality of hybridized complexes; and
binding the hybridized complexes to an active binding surface for imaging thereof.