Multi-stage, multiplexed target isolation and processing from heterogeneous populations

A substrate-isolation platform, comprising:

• a microfluidic chip, comprising a plurality of processing units, each processing unit comprising:
• an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve;
• a magnet adjacent the microfluidic chip, wherein relative positioning of the magnet and the microfluidic chip is variable;
• and
• a valve control capable of actuating certain ones of the controllable blocking valves in response to a control signal.

A method of integrated subtype purification and RNA-Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising:

• an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve;
• and a movable magnet adjacent the microfluidic chip, the method comprising:
• providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;
• providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell-type bind to the first magnetic beads;
• applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;
• blocking fluid communication between the second chamber and the first chamber;
• washing the first chamber;
• unblocking fluid communication between the second chamber and the first chamber;
• releasing the cells from the first magnetic beads;
• removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;
• blocking fluid communication between the second chamber and the first chamber;
• lysing the cells in the second chamber;
• capturing target substrates from the lysed cells using dynamically altered magnetic beads;
• applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber;
• mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;
• cycling through a range of temperatures to create a PCR product;
• and
• applying DNA-binding beads to the PCR product to clean up DNA.

A kit comprising an integrated subtype purification and RNA-Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising:

• an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve;
• and a movable magnet adjacent the microfluidic chip, the method comprising:
• providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;
• providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell-type bind to the first magnetic beads;
• applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;
• blocking fluid communication between the second chamber and the first chamber;
• washing the first chamber;
• unblocking fluid communication between the second chamber and the first chamber;
• releasing the cells from the first magnetic beads;
• removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;
• blocking fluid communication between the second chamber and the first chamber;
• lysing the cells in the second chamber;
• capturing target substrates from the lysed cells using dynamically altered magnetic beads;
• applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber;
• mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;
• cycling through a range of temperatures to create a PCR product;
• and
• applying DNA-binding beads to the PCR product to clean up DNA.