DROPLET-BASED AFFINITY ASSAYS

• US 20130252262A1
• Filed: 05/09/2013
• Published: 09/26/2013
• Est. Priority Date: 04/13/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Srinivasan, Vijay, Pamula, Vamsee K., Pollack, Michael G., Fair, Richard B.
• Granted Patent: US 9,476,856 B2
• Time in Patent Office: Days
• US Class Current: 435/7.72
• CPC Class Codes: G01N 27/72 by investigating magnetic variables
• CPC Class Codes: G01N 27/745 for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 33/54326 Magnetic particles

Abstract

A method of detecting a target analyte in a sample, including executing electrowetting-mediated droplet operations and thereby: combining one or more immunoassay reagent droplets comprising magnetically-responsive beads having affinity for the target analyte with one or more sample droplets potentially comprising the target analyte to yield a first combined droplet; beginning with the combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a first washed droplet comprising the washed magnetically responsive beads; and combining the droplet comprising the washed magnetically responsive beads with a droplet comprising a reporter molecule having affinity for target analyte captured on the magnetically-responsive beads to yield a second combined droplet; beginning with the second combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a second washed droplet comprising the washed magnetically responsive beads; detecting a signal from the second washed droplet which corresponds to the presence, absence and/or quantity of the analyte in the sample.

Claims

1. A method of detecting a target analyte in a sample, the method comprising:

(a) executing electrowetting-mediated droplet operations and thereby;

(i) combining (A) one or more immunoassay reagent droplets comprising magnetically-responsive beads having affinity for the target analyte with (B) one or more sample droplets potentially comprising the target analyte to yield (C) a first combined droplet;

(ii) beginning with the combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a first washed droplet comprising the washed magnetically responsive beads; and

(iii) combining (A) the droplet comprising the washed magnetically responsive beads with (B) a droplet comprising a reporter molecule having affinity for target analyte captured on the magnetically-responsive beads to yield (C) a second combined droplet;

(iv) beginning with the second combined droplet, effecting a droplet-based washing protocol to wash the magnetically-responsive beads to yield a second washed droplet comprising the washed magnetically responsive beads;

(b) detecting a signal from the second washed droplet which corresponds to the presence, absence and/or quantity of the analyte in the sample.

2. The method of claim 1 wherein the detecting step comprises detecting a signal from the second washed droplet.

3. The method of claim 1 wherein the signal measured corresponds to unoccupied binding sites on the one or more magnetically responsive beads.

4. The method of claim 1 wherein the signal measured corresponds to occupied binding sites on the one or more magnetically responsive beads.

5. The method of claim 1 wherein the reporter molecule comprises a labeled antibody.

6. The method of claim 5 wherein the labeled antibody comprises a radioactive isotope.

7. The method of claim 1 wherein the reporter molecule comprises an antibody conjugated to an enzyme capable of catalyzing a detectable reaction.

8. The method of claim 7 wherein the detectable reaction result in a colorimetric, fluorescent and/or luminescent property or signal.

9. The method of claim 1 wherein the one or more sample droplets comprise a substance selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes.

10. The method of claim 1 wherein the method further comprises providing the sample droplet by conducting electrowetting-mediated dispensing of the sample droplet on the droplet actuator.

11. The method of claim 1 wherein the signal comprises an electrochemical property of a droplet on a droplet microactuator.

12. The method of claim 11 wherein the electrochemical property comprises an amperometric, potentiometric and/or conductometric property of a droplet on the droplet microactuator.

13. The method of claim 1 wherein the signal comprises an optical property of a droplet on a droplet microactuator.

14. The method of claim 13 wherein the optical property comprises absorbance, chemiluminescence and/or fluorescence.

15. The method of claim 13 wherein the optical property comprises Raman spectra.

16. The method of claim 1 wherein the signal comprises a physical parameter of a droplet on a droplet microactuator.

17. The method of claim 16 wherein the physical parameter comprises temperature.

18. The method of claim 16 wherein the physical parameter comprises emission of radioactive particles.