Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest

• US 10,450,598 B2
• Filed: 09/08/2016
• Issued: 10/22/2019
• Est. Priority Date: 09/11/2015
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Ho, Hwai-En, Smith, Gregory F., Dhopeshwarkar, Rahul R.
• Primary Examiner(s): Dejong, Eric S
• Application Number:
  US15/259,310
• Publication Number:
  US 20170073729A1
• Time in Patent Office: 1,139 Days
• Field of Search: None
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

Method includes providing a droplet actuator having a droplet-operations gap and a plurality of electrodes positioned along the droplet-operations gap. The method also includes positioning an input droplet in the droplet-operations gap. The input droplet has a starting concentration of a substance-of-interest. The method also includes conducting droplet operations within the droplet-operations gap using the electrodes to generate discrete dilution droplets that are formed from the input droplet. The dilution droplets and a remainder of the input droplet form a droplet set. At least two of the dilution droplets in the droplet set having different concentrations of the substance-of-interest. The method also includes combining a select number of the droplets from the droplet set to form an output droplet having a modified concentration that is substantially equal to a designated target concentration.

Claims

1. A method comprising:

providing a droplet actuator having a droplet-operations gap and a plurality of electrodes positioned along the droplet-operations gap;

positioning an input droplet in the droplet-operations gap, the input droplet having a starting concentration of a substance-of-interest;

conducting droplet operations within the droplet-operations gap using the electrodes to generate discrete dilution droplets that are formed from the input droplet, wherein the dilution droplets and a remainder of the input droplet form a droplet set, at least two of the dilution droplets in the droplet set having different concentrations of the substance-of-interest; and

combining a select number of the droplets from the droplet set to form an output droplet having a modified concentration that is substantially equal to a designated target concentration.

2. The method of claim 1, wherein each of the dilution droplets is formed by combining a first volume of a dilution liquid and a second volume of another droplet, the other droplet being the input droplet or one of the other dilution droplets.

3. The method of claim 2, wherein the first and second volumes are equal volumes such that the concentration of each dilution droplet is about half the concentration of the other droplet.

4. The method of claim 1, wherein the dilution droplets that are selected to be combined are based on at least one of:

(a) a designated number of dilution operations;

(b) a designated number of droplets available for combining;

(c) a designated number of droplets to be combined together to form the output droplet;

(d) the starting concentration of the input droplet;

or (e) the target concentration of the output droplet.

5. The method of claim 1, further comprising identifying the select number of the droplets to be combined based on a dilution fraction (DF), the DF being equal to:

(C_O×

k×

2^d)÷

C_I;

wherein d is the number of dilution operations, k is the number of droplets to be combined to form the output droplet, C_I is the concentration of the input droplet, and C_O is the target concentration of the output droplet.

6. The method of claim 5, wherein identifying the select number of droplets to be combined includes:

(a) identifying an integer that is approximately equal to the DF and converting the integer into an n-bit binary number, wherein n is the number of dilution droplets available for combining;

(b) counting a number of active digits in the n-bit binary number;

(c) determining whether the number of active digits is equal to k;

wherein, if the number of active digits is equal to k, the method includes correlating the active digits of the n-bit binary number to the droplets in the droplet set thereby identifying the droplets to be combined;

wherein, if the number of active digits does not equal k, the method includes repeating (a)-(c) for a different integer.

7. The method of claim 1, further comprising identifying the select number of the droplets to be combined using a lookup table, the lookup table including (a) a range of designated concentrations for the input droplet and (b) a range of designated concentrations for the output droplet.

8. The method of claim 7, wherein the lookup table also includes at least one of:

(a) a range of numbers that represent the number of dilution operations that can be performed;

(b) a range of numbers that represent the number of droplets available for mixing;

(c) a range of numbers that represent the number of droplets to be combined together to form the output droplet.

9. The method of claim 8, wherein the lookup table is populated based on a dilution fraction (DF), the DF being equal to:

(C_O×

k×

2^d)÷

C_I;

wherein d is the number of dilution operations, k is the number of droplets to be combined together to form the output droplet, C_I is the concentration of the input droplet, and C_O is the target concentration of the output droplet.

10. The method of claim 1, further comprising receiving user inputs that designate at least one of (a) a number of times dilution is performed;

(b) a number of droplets available for mixing;

(c) a number of droplets to be combined together to form the output droplet;

(d) the starting concentration of the input droplet;

or (e) the target concentration of the output droplet.

11. The method of claim 1, further comprising determining the target concentration by at least one of receiving user inputs about the input droplet or analyzing the input droplet on the droplet actuator.

12. A system comprising:

a droplet actuator having a droplet-operations gap and a plurality of electrodes positioned along the droplet-operations gap, the droplet-operations gap configured to receive an input droplet, the input droplet having a starting concentration of a substance-of-interest; and

a controller operably coupled to the electrodes of the droplet actuator, the controller configured to control the electrodes to;

conduct droplet operations within the droplet-operations gap using the electrodes to generate discrete dilution droplets that are formed from the input droplet, wherein the dilution droplets and a remainder of the input droplet form a droplet set, at least two of the dilution droplets in the droplet set having different concentrations of the substance-of-interest; and

combining a select number of the droplets from the droplet set to form an output droplet, the output droplet having a modified concentration of the substance-of-interest that is substantially equal to a designated target concentration.

13. The system of claim 12, wherein the modified concentration is substantially equal to the target concentration if the modified concentration is within 30% of the target concentration.

14. The system of claim 12, wherein the system is configured to perform a library preparation assay using the output droplet.

15. A method comprising:

loading sample droplets into a droplet actuator, the droplet actuator having a droplet-operations gap and a plurality of electrodes positioned along the droplet-operations gap, the sample droplets being separated from one another in the droplet-operations gap and including a substance-of-interest; and

determining respective concentrations of the substance-of-interest in the sample droplets;

adjusting the respective concentration of at least one of the sample droplets so that each of the respective concentrations of at least a plurality of the sample droplets is substantially equal to a target concentration, wherein adjusting the respective concentration of a corresponding sample droplet includes;

conducting droplet operations within the droplet-operations gap using the electrodes to generate discrete dilution droplets that are formed from a corresponding sample droplet, wherein the dilution droplets and a remainder of the corresponding sample droplet form a droplet set, at least two of the dilution droplets in the droplet set having different concentrations of the sub stance-of-interest;

combining a select number of the droplets from the droplet set to form an output droplet having a modified concentration, the select number of the droplets being based on the respective concentration of the corresponding sample droplet and the target concentration.

16. The method of claim 15, wherein the substance-of-interest comprises nucleic acids.

17. The method of claim 16, further comprising performing a sequencing-by-synthesis assay using the output droplet and the other sample droplets or outlet droplets that are based on the other sample droplets.

18. The method of claim 16, wherein the sample droplets are from different sources, the nucleic acids of each sample droplet being tagged to indicate the corresponding source.

19. The method of claim 15, further comprising determining the target concentration, wherein determining the target concentration includes identifying the lowest respective concentration among the sample droplets, the target concentration being based on the lowest respective concentration.

20. The method of claim 15, further comprising determining the target concentration, wherein determining the target concentration includes receiving user inputs that confirm a suggested target concentration.