EWOD system and methods to increase dynamic range for digital nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-11198130-B2
Application number	US-201816014006-A
Country	US
Kind code	B2
Filing date	Jun 21, 2018
Priority date	Jun 21, 2018
Publication date	Dec 14, 2021
Grant date	Dec 14, 2021

Abstract

A method of digital quantification of a species in an EWOD device includes inputting a sample volume and a diluent volume into the EWOD device; performing an electrowetting operation to generate a first sample droplet from the sample volume; performing an amplification process on the first sample droplet and measuring a turn-on value for the sample droplet; comparing the measured turn-on value to a target turn-on value for digital quantification; calculating a dilution factor based on the comparison of the measured and target turn-on values; performing an electrowetting operation to extract a second sample droplet from the sample volume; performing an electrowetting operation to dilute the second sample droplet with the diluent volume by the dilution factor to form a diluted second sample droplet; and performing a digital quantification on the diluted second sample droplet to quantify an initial concentration of the species in the sample volume.

First claim

What is claimed is:

1. A method of performing a digital quantification of a species in an electrowetting on dielectric (EWOD) device comprising the steps of:
inputting a sample volume into the EWOD device;
inputting a diluent volume into the EWOD device;
performing an electrowetting operation to generate a first sample droplet from the sample volume;
performing an amplification process on the first sample droplet within the EWOD device;
measuring a turn-on value for the sample droplet, wherein the turn-on value comprises a point at which fluorescence crosses above a threshold value relative to a baseline fluorescence;
comparing the measured turn-on value of the sample droplet to a target turn-on value for digital quantification;
calculating a dilution factor based on the comparison of the measured turn-on value of the sample droplet to the target turn-on value;
performing an electrowetting operation to extract a second sample droplet from the sample volume;
performing an electrowetting operation to dilute the second sample droplet with the diluent volume in accordance with the dilution factor to form a diluted second sample droplet;
performing a digital quantification on the diluted second sample droplet to quantify an initial concentration of the species in the sample volume; and
determining an efficiency of reaction of the amplification process of the first sample droplet to calculate the dilution factor.

2. The method of claim 1 , wherein the first sample droplet is moved by electrowetting forces into a sample preparation zone, and further is moved by electrowetting forces into a first amplification zone in which the amplification of the species of interest on the first sample droplet is performed, and the first amplification zone is separate from the sample preparation zone.

3. The method of claim 2 , further comprising using electrowetting forces to dilute the first sample droplet with a portion of the diluent volume when the first sample droplet is in the sample preparation zone to form an initial diluted sample droplet, and moving the initial diluted sample droplet into the first amplification zone to perform amplification on the initial diluted sample droplet.

4. The method of claim 3 , wherein performing the digital amplification comprises using electrowetting forces to split the diluted second droplet into partitions that are moved into a digital amplification zone that is separate from the sample preparation zone and the first amplification zone.

5. The method of claim 1 , wherein the EWOD device includes a fluorescence measurement apparatus, and the turn-on value of the sample droplet is determined using a fluorescence intensity curve.

6. The method of claim 1 , wherein performing the digital quantification includes performing a normalization process by detecting fluorescence relative to fluorescence of a reference dye contained in the sample volume.

7. The method of claim 1 , wherein performing the digital quantification includes:
partitioning the diluted sample droplet into a plurality of partitions;
performing an amplification process on the plurality of partitions;
counting a number of positive partitions and a number of negative partitions; and
using Poisson statistics to quantify an initial concentration of the species in the sample volume.

8. The method of claim 7 , further comprising adding a scaling factor to ensure a proportion of positive partitions is between 0.01%-90%.

9. The method of claim 1 , wherein determining the efficiency of reaction comprises the steps of:
performing an electrowetting operation to extract another sample droplet from the sample volume;
performing amplification on the another sample droplet within the EWOD device;
measuring a second turn-on value for the another sample droplet;
determining an efficiency of reaction using the turn-on value and the second turn-on value; and
calculating the dilution factor using the efficiency of reaction applied to the comparison of the measured turn-on value of the sample droplet to the target turn-on value.

10. The method of claim 1 , further comprising:
inputting a plurality of target input volumes into the EWOD device having respective primers corresponding to a plurality of respective targets in the sample volume;
performing an electrowetting operation to extract a respective daughter sample droplet from the sample volume corresponding to each of the plurality of target input volumes;
performing an electrowetting operation to dilute the daughter droplets with the respective target input volumes in accordance with the dilution factor to form a plurality of respective diluted sample/target volume combinations; and
performing the amplification process on the diluted sample/target volume combinations to quantify an initial concentration of the respective targets on the species in the sample volume.

11. The method of claim 10 , further comprising:
inputting a quantification volume into the EWOD device;
dispensing the first sample droplet and a quantification droplet from the quantification volume into a zone of the EWOD device and mix said droplets together to form an amplifying droplet;
performing the amplification process on the amplifying droplet;
measuring a turn-on value for the amplifying droplet;
comparing the measured turn-on value of the amplifying droplet to the target turn-on value; and
calculating the dilution factor based on the comparison of the measured turn-on value of the amplifying droplet to the target turn-on value.

12. The method of claim 1 , wherein the sample volume contains a protein species.

13. The method of claim 1 , wherein the sample volume contains a cell species.

14. A method of performing a digital quantification of a species in an electrowetting on dielectric (EWOD) device comprising the steps of:
inputting a sample volume into the EWOD device;
inputting a diluent volume into the EWOD device;
performing an electrowetting operation to generate a first sample droplet from the sample volume;
performing an amplification process on the first sample droplet within the EWOD device;
measuring a turn-on value for the sample droplet;
comparing the measured turn-on value of the sample droplet to a target turn-on value for digital quantification;
calculating a dilution factor based on the comparison of the measured turn-on value of the sample droplet to the target turn-on value;
performing an electrowetting operation to extract a second sample droplet from the sample volume;
performing an electrowetting operation to dilute the second sample droplet with the diluent volume in accordance with the dilution factor to form a diluted second sample droplet; and
performing a digital quantification on the diluted second sample droplet to quantify an initial concentration of the species in the sample volume
wherein the sample volume contains a nucleic acid species, the EWOD device includes a plurality of thermal control elements, and the amplification process performed on the first sample droplet and/or the digital amplification is a polymerase chain reaction (PCR) nucleic acid amplification; and
wherein the turn-on value is a threshold cycle value (Ct) for the sample droplet.

15. A method of performing a digital quantification of a species in an electrowetting on dielectric (EWOD) device comprising the steps of:
inputting a sample volume into the EWOD device;
inputting a diluent volume into the EWOD device;
performing an electrowetting operation to generate a first sample droplet from the sample volume;
performing an amplification process on the first sample dro

Assignees

• Sharp Life Science Eu Ltd

Inventors

• Dothie Pamela Ann
• Anderson Sally
• Roberts Philip Mark Shryane

Classifications

• B01F33/30351

  using hydrophilic/hydrophobic surfaces · CPC title

• B01F33/3021

  the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets · CPC title

• B01L3/50273

  characterised by the means or forces applied to move the fluids · CPC title

• B01L3/502792 Primary

  for moving individual droplets on a plate, e.g. by locally altering surface tension · CPC title

• B01L2400/0427

  Electrowetting · CPC title