# US 2019/0169700 A1 · Methods and Systems for Detecting Biological Components

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- Number: US 2019/0169700 A1
- Document ID: US20190169700A1
- Status: Published application
- Kind: A1
- Application number: 16/279,905
- Filed: 2019-02-19
- Published: 2019-06-06
- Earliest claimed: 2012-08-13
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Adam R. Abate; Dennis Jay Eastburn; Adam R. Sciambi
- Web page: https://patentgrep.com/patent/US20190169700A1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- B01F 23/41: Emulsifying
- B01F 25/4335: {Mixers with a converging-diverging cross-section}
- B01F 33/3011: using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
- B01F 33/3031: using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/686: Polymerase chain reaction [PCR]
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/0883: Serpentine channels
- B01L 2300/1822: using Peltier elements
- B01L 2400/0415: electrical forces, e.g. electrokinetic
- B01L 2400/0487: fluid pressure, pneumatics
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

## Claims

### Claim at position 1

-146. (canceled)

### Claim 147

A method of synthesizing a target polynucleotide, the method comprising:
contacting a polynucleotide-containing component from a sample with lysis reagents in a droplet, the lysis reagents comprising an enzyme having protease activity, wherein the droplet is encapsulated with an immiscible carrier fluid;
moving the droplet into a collection reservoir;
incubating the droplet in the collection reservoir for a first duration and then inactivating the enzyme having protease activity;
adding to the droplet a nucleic acid synthesis reagent to form a nucleic acid synthesis droplet in the immiscible carrier fluid to form a nucleic acid droplet; and
synthesizing the target polynucleotide within the nucleic acid synthesis droplet.

### Claim 148 (depends on 147)

The method of claim 147, wherein the step of contacting a polynucleotide-containing component from a sample with lysis reagents in a droplet is implemented on a first device.

### Claim 149 (depends on 147)

The method of claim 147, wherein the collection reservoir is associated with a second device.

### Claim 150 (depends on 149)

The method of claim 149, wherein the first device and the second device are not integrated.

### Claim 151 (depends on 147)

The method of claim 147, wherein the first duration is a period longer than necessary for lysis.

### Claim 152 (depends on 147)

The method of claim 147, wherein the step of incubating the droplet in a collection reservoir further comprises incubating the lysate droplet and the enzyme having protease activity in the collection reservoir at a temperature sufficient to inactivate the enzyme having protease activity.

### Claim 153 (depends on 147)

The method of claim 147, wherein the step of adding to the droplet a nucleic acid synthesis reagent further comprises adding a plurality of nucleic acid synthesis reagents.

### Claim 154 (depends on 147)

The method of claim 147, wherein the nucleic acid synthesis droplet has a volume of 0.001 to 1000 picoliters.

### Claim 155 (depends on 147)

The method of claim 147, wherein nucleic acid synthesis droplet has a diameter of between 0.1 microns to 1000 microns.

### Claim 156 (depends on 147)

The method of claim 147, wherein step of adding a nucleic acid synthesis reagent to the droplet further comprises:
(i) contacting the lysate reagents with a continuous stream of fluid comprising the nucleic acid synthesis reagent, and
(ii) forming the nucleic acid synthesis droplet from a portion of the continuous stream of fluid and the droplet.

### Claim 157 (depends on 147)

The method of claim 147, further comprising detecting the target polynucleotide by determining a sequence of a nucleic acid synthesis product of the nucleic acid synthesis droplet or by forming a double-emulsion comprising the nucleic acid synthesis droplet within an outer droplet, and sorting the double-emulsion based on at least one of droplet size and fluorescence.