# US 10,309,927 B2 · Methods of conducting biochemical reactions while reducing reactive molecular species during electrowetting

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- Number: US 10,309,927 B2
- Document ID: US10309927B2
- Status: Granted
- Kind: B2
- Application number: 15/093,529
- Filed: 2016-04-07
- Published: 2016-10-13
- Granted: 2019-06-04
- Earliest claimed: 2015-04-10
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Rigo Pantoja; Rachel M. Schowalter; Jimmy Perrott; Cyril Delattre; Allen E. Eckhardt
- Web page: https://patentgrep.com/patent/US10309927B2

## CPC

- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- G01N 27/44743: {Introducing samples}
- B01L 2400/0421: electrophoretic flow
- B01L 2400/0427: Electrowetting
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Some embodiments disclosed herein provide methods of reducing reactive molecular species in a reaction carried out in a digital fluidics device having one or more electrodes by using a reaction mixture comprising an electron acceptor additive. In some embodiments, the presence of the electron acceptor additive reduces the presence of reactive molecular species in the reaction mixture. In some embodiments, the presence of the electron acceptor additive reduces the formation of gas bubbles in the digital fluidic device. Therefore, in preferred embodiments, the electron acceptor additive does not form a gas when reduced.

## Claims

### Claim 1

A method of reducing reactive molecular species in a reaction carried out in a digital fluidics device having one or more electrodes, the method comprising:
providing a reaction mixture comprising one or more reagents and an electron acceptor additive;
forming a reaction droplet from the reaction mixture; and
activating the one or more electrodes to conduct a reaction, wherein the presence of the electron acceptor additive reduces the presence of reactive molecular species in the reaction mixture, wherein the reduction in the formation of reactive molecular species reduces the formation of bubbles.

### Claim 2 (depends on 1)

The method of claim 1, wherein the electron acceptor additive comprises a redox potential lower than 830 mV.

### Claim 3 (depends on 1)

The method of claim 1, wherein the electron acceptor additive comprises phenazine ethosulfate (PES), phenazine methosulfate (PMS), or a combination thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the electron acceptor additive prevents the electrolysis of water.

### Claim 5 (depends on 1)

The method of claim 1, wherein the electron acceptor additive is not a reactant in the reaction.

### Claim 6 (depends on 1)

The method of claim 1, wherein the electron acceptor additive does not form a gas when reduced.

### Claim 7 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises low conductivity in a range of about 2.5±0.2 μS/cm to about 5±0.8 μS/cm.

### Claim 8 (depends on 1)

The method of claim 1, wherein the reaction mixture comprises low pH of about pH 2.0 to pH 7.0.

### Claim 9 (depends on 1)

The method of claim 1, wherein activating the one or more electrodes comprises selecting droplet electrowetting parameters that result in a reduction in the formation of reactive molecular species.

### Claim 10 (depends on 9)

The method of claim 9, wherein the droplet electrowetting parameters comprise one or more of droplet size, transport rate, and electrode exposure.

### Claim 11 (depends on 10)

The method of claim 10, wherein droplet size is less than 8 digital units.

### Claim 12 (depends on 1)

The method of claim 1, wherein the reaction is a sequencing-by-synthesis (SBS) reaction.

### Claim 13 (depends on 1)

The method of claim 1, wherein the reaction is a nucleic acid amplification reaction.

### Claim 14 (depends on 1)

The method of claim 1, wherein the reactive molecular species comprise reactive oxygen species (ROS) or hypochlorite.

### Claim 15 (depends on 1)

The method of claim 1, further comprising surrounding the reaction droplet with an immiscible fluid.

### Claim 16 (depends on 15)

The method of claim 15, wherein the immiscible fluid comprises a modified polysiloxane polymer.

### Claim 17

A method of reducing reactive molecular species in a reaction carried out in a digital fluidics device having one or more electrodes, the method comprising:
providing a reaction droplet;
surrounding the reaction droplet with an immiscible fluid comprising a modified polysiloxane polymer; and
activating the one or more electrodes to conduct a reaction, wherein the presence of the polysiloxane polymer reduces the presence of reactive molecular species in the reaction droplet, wherein the reduction in the formation of reactive molecular species reduces the formation of bubbles.

### Claim 18 (depends on 17)

The method of claim 17, wherein the modified polysiloxane polymer has the following formula:
wherein m=1-300, n=1-300, and p=1-50.

### Claim 19 (depends on 18)

The method of claim 18, wherein the modified polysiloxane polymer comprises POLY(3,3,3-TRIFLUOROPROPYLMETHYLSILOXANE), HYDROXYPROPYLENEOXYPROPYL)METHYLSILOXANE-DIMETHYLSILOXANE COPOLYMER, 1,3-BIS(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL) TETRAMETHYLDISILOXANE, or a combination thereof.

### Claim 20 (depends on 17)

The method of claim 17, wherein the modified polysiloxane polymer has the following formula:
wherein each R1 is, independently, hydrogen, C1-8 alkyl, C6-30 aryl, or C1-15 alkyl-substituted C6-30 aryl;
R2 is, independently, R1, R3, R4, R5, or R6;
R3 is, independently, a C1-10 fluoroalkyl or C1-15 fluoroalkyl-substituted C6-30 aryl, where fluoroalkyl is defined as having anywhere from 1 fluorine atom to complete fluorination;
R4 is, independently, a polyalkylene glycol moiety, where the alkylene groups are, independently, C2-4, and the number of repeat units in the moiety is between 1 and 1000, more typically, between 1 and 150, and still more typically, between 1 and 50 repeat units, wherein specific polyalkylene glycol moieties include polyethylene glycol, polypropylene glycol, and copolymers thereof;
R5 is, independently, a dye selected from the group consisting of erioglaucine, Nile blue, methylene blue, methyl viologen, methyl brilliant green, popop brilliant green, caffeine dye, proton sponge dye, and DDT black, linked to the polymer at any position;
R6 is an electron acceptor;
m is a whole number from 0 to about 300,
n is a whole number from 0 to about 300,
o is a whole number from 0 to about 300, and
p is a whole number from 0 to about 300,
wherein at least one of m, n, o, and p is not 0.

### Claim 21 (depends on 20)

The system of claim 20, wherein each R1 is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, isobutyl, isoamyl, or t-amyl.

### Claim 22

A system for conducting a reaction in the presence of an activated electrode while reducing the formation of reactive molecular species, wherein the reduction in the formation of reactive molecular species reduces the formation of bubbles, the system comprising:
a fluidics cartridge comprising an electrowetting array having a plurality of electrodes, wherein the fluidics cartridge comprises a substrate having a hydrophobic coating having a thickness of greater than about 1,000 nm.

### Claim 23 (depends on 22)

The system of claim 22, further comprising an immiscible fluid, wherein the immiscible fluid comprises a modified polysiloxane polymer.

### Claim 24 (depends on 23)

The system of claim 23, wherein the modified polysiloxane polymer has the following formula:
wherein m=1-300, n=1-300, and p=1-50.

### Claim 25 (depends on 23)

The system of claim 23, wherein the modified polysiloxane polymer comprises POLY(3,3,3-TRIFLUOROPROPYLMETHYLSILOXANE), HYDROXYPROPYLENEOXYPROPYL)METHYLSILOXANE-DIMETHYLSILOXANE COPOLYMER, 1,3-BIS(TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL) TETRAMETHYLDISILOXANE, or a combination thereof.

### Claim 26 (depends on 23)

The system of claim 23, wherein the modified polysiloxane polymer has the following formula:
wherein each R1 is, independently, hydrogen, C1-8 alkyl, C6-30 aryl, or C1-15 alkyl-substituted C6-30 aryl;
R2 is, independently, R1, R3, R4, R5, or R6;
R3 is, independently, a C1-10 fluoroalkyl or C1-15 fluoroalkyl-substituted C6-30 aryl, where fluoroalkyl is defined as having anywhere from 1 fluorine atom to complete fluorination;
R4 is, independently, a polyalkylene glycol moiety, where the alkylene groups are, independently, C2-4, and the number of repeat units in the moiety is between 1 and 1000, more typically, between 1 and 150, and still more typically, between 1 and 50 repeat units, wherein specific polyalkylene glycol moieties include polyethylene glycol, polypropylene glycol, and copolymers thereof;
R5 is, independently, a dye selected from the group consisting of erioglaucine, Nile blue, methylene blue, methyl viologen, methyl brilliant green, popop brilliant green, caffeine dye, proton sponge dye, and DDT black, linked to the polymer at any position;
R6 is an electron acceptor;
m is a whole number from 0 to about 300,
n is a whole number from 0 to about 300,
o is a whole number from 0 to about 300, and
p is a whole number from 0 to about 300,
wherein at least one of m, n, o, and p is not 0.

### Claim 27 (depends on 26)

The system of claim 26, wherein each R1 is independently hydrogen, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t-butyl, isobutyl, isoamyl, or t-amyl.