Bead incubation and washing on a droplet actuator
Methods are provided for separating magnetically responsive beads from a droplet in a droplet actuator. Droplet operations electrodes and a magnet are arranged in a droplet actuator to manipulate a bead-containing droplet and position it relative to a magnetic field region that attracts the magnetically responsive beads. The droplet operations electrodes are operated to control the droplet shape and transport it away from the magnetic field region to form a concentration of beads in the droplet. The continued transport of the droplet away from the magnetic field causes the concentration of beads to break away from the droplet to yield a small, concentrated bead-containing droplet immobilized by the magnet.
1. A method of separating magnetically responsive beads from a droplet, the method comprising:
(a) providing a droplet actuator comprising:
(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and
(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet;
(b) positioning a droplet having magnetically responsive beads therein within the first region of the magnetic field of the magnet to cause the magnetically responsive beads to be attracted to the magnet, and activating the droplet operations surface to cause the droplet to be circular in shape;
(c) operating the droplet operations surface to cause the droplet to move away from the first region of the magnetic field to form a concentration of magnetically responsive beads in the droplet, and the droplet operations surface being operated to cause the droplet to be transported away from the magnet one droplet operations electrode at a time, to cause the geometry of the droplet to be distorted; and
(d) continuing to transport the droplet away from the magnet to cause the concentration of magnetically responsive beads to break away from the droplet to result in a relatively small and highly concentrated magnetically responsive bead droplet left behind and held immobilized by the magnet.
2. The method of claim 1 , wherein in (c) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent to the magnet.
3. The method of claim 1 , wherein in (b) the droplet is positioned at a droplet operations electrode within the first region of the magnetic field of the magnet.
4. The method of claim 1 , wherein in (c) the droplet is caused to move from the droplet operations electrode within the first region of the magnetic field partially to an adjacent droplet operations electrode away from the magnet.
5. The method of claim 1 , wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target sub stance.
6. The method of claim 5 , wherein the target substance is at least one of a cell, a protein, a nucleic acid, and an antigen.
7. The method of claim 5 , wherein the target substance is a cytokine.
8. The method of claim 1 , wherein the droplet comprises, by volume, one part magnetically responsive beads to one part sample.
9. The method of claim 1 , wherein the droplet comprises, by volume, one part sample plus magnetically responsive beads to one part secondary reporter antibodies.
10. The method of claim 1 , wherein the magnet is positioned such that three droplet operations electrodes are within the magnetic field of the magnet.
11. The method of claim 1 , wherein the magnet is one of a permanent magnet and an electromagnet.
12. The method of claim 1 , wherein the droplet transport involves electrowetting.
13. The method of claim 1 , wherein the droplet transported has a footprint approximately two times the area of any one of the droplet operations electrodes.
14. The method of claim 1 , wherein the droplet transported has a footprint approximately three times the area of any one of the droplet operations electrodes.
15. The method of claim 1 , wherein the magnetically responsive beads are coated with a primary antibody with an affinity for a specific target antigen.
16. A method of separating magnetically responsive beads from a droplet, the method comprising:
(a) providing a droplet actuator comprising:
(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and
(ii) a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet;
(b) positioning an elongated droplet containing magnetically responsive beads over at least three active droplet operations electrodes that include an intermediate droplet operation electrode, the at least three droplet operations electrodes positioned within the first region of the magnetic field of the magnet to cause the magnetically responsive beads to be attracted to the magnet;
(c) operating the droplet operations surface to cause the droplet to move away from the first region of the magnetic field to form a concentration of magnetically responsive beads in the droplet, and the droplet operations surface being operated to cause the droplet to be transported away from the magnet one droplet operations electrode at a time, to cause the geometry of the droplet to be distorted; and
(d) deactivating an intermediate droplet operations electrode to split the droplet into a supernatant droplet and a smaller droplet containing the beads held immobilized by the magnet.
17. The method of claim 16 , wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.
18. The method of claim 17 , wherein the target substance is at least one of a cell, a protein, a nucleic acid, and an antigen.
19. The method of claim 17 , wherein the target substance is a cytokine.
20. The method of claim 16 , wherein in (c) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent to the magnet.