# US 10,369,569 B2 · Method of identifying droplets in a stack and an associated sequencer

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- Number: US 10,369,569 B2
- Document ID: US10369569B2
- Status: Granted
- Kind: B2
- Application number: 15/574,005
- Filed: 2017-07-14
- Published: 2018-09-20
- Granted: 2019-08-06
- Earliest claimed: 2016-07-14
- Assignees: Base4 Innovation Ltd
- Applicants: Base4 Innovation Ltd
- Inventors: Cameron Alexander Frayling; Thomas Henry Isaac
- Web page: https://patentgrep.com/patent/US10369569B2

## CPC

- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- B01L 3/0268: {using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries}
- C12Q 1/6869: Methods for sequencing
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- B01J 2219/00367: capillary
- B01J 2219/00576: fluorophore
- B01J 2219/00702: Processes involving means for analysing and characterising the products
- B01L 2200/0642: Filling fluids into wells by specific techniques
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2300/0627: Sensor or part of a sensor is integrated
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- C40B 60/12: for screening libraries
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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
- 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)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Disclosed is a method of identifying the contents of individual droplets in a droplet stream each droplet containing fluorophores in an initial non-fluorescing state characterized by the steps of introducing the droplets one-by-one into at least one open-ended tube to create a stack of droplets therein; activating the fluorophores within the droplets to cause them to fluoresce; releasing each droplet in the droplet stack in turn from the tube and detecting along the major axis of the tube fluorescence associated with each droplet as it emerges. Also disclosed is a method suitable for sequencing a biopolymer characterized by the steps of (1) progressively digesting the biopolymer into an ordered stream of its constituent monomers; (2) converting the stream of monomers into a corresponding stream of monomer-containing aqueous droplets each droplet additionally containing a probe capable of (a) capturing the monomer and (b) thereafter being digested to release an unqueched fluorophore characteristic of the captured monomer; (3) introducing the stream of droplets created in step (2) into an inlet end of at least one open-ended tube to create a stack of droplets therein and (4) releasing each droplet in turn from an outlet end of the tube(s) and detecting fluorophores in each droplet as each droplet emerges. The method may be used in a corresponding apparatus for sequencing a biopolymer such as a nucleic acid or protein.

## Claims

### Claim 1

A method of identifying the contents of individual droplets in a droplet stream wherein each droplet contains fluorophores, the method comprising the steps of: (a) introducing the droplets one-by-one into at least one open-ended tube to create a stack of droplets therein; (b) releasing each droplet in the droplet stack in turn from the tube; and (c) detecting along a major axis of the tube fluorescence associated with each droplet as each droplet emerges.

### Claim 2 (depends on 1)

The method of claim 1, wherein the droplets are introduced into an inlet end of the tube and released from an outlet end.

### Claim 3 (depends on 1)

The method of claim 1, wherein the tube is vertically aligned and each droplet is released from the bottom of the droplet stack via the outlet end.

### Claim 4 (depends on 1)

The method of claim 1, wherein the fluorophores in the droplets are in an initial non-fluorescing state, and wherein the method further comprises a step of activating the fluorophores whilst the droplets are located within the droplet stack.

### Claim 5 (depends on 1)

The method of claim 1, wherein the fluorophores are associated with a probe molecule which is selective for and characteristic of an analyte molecule which is to be identified.

### Claim 6 (depends on 1)

The method of claim 1, further comprising an additional step of sorting or characterising the droplets by the fluorescence signal detected.

### Claim 7 (depends on 1)

The method of claim 1, wherein a negative pressure is applied across the tube whilst the tube is being filled with droplets from the inlet end.

### Claim 8 (depends on 1)

The method of claim 1, wherein the tube has an internal diameter that is less than twice the diameter of the droplets.

### Claim 9 (depends on 1)

The method of claim 1, wherein the droplets in the droplet stack are suspended in an immiscible solvent.

### Claim 10 (depends on 1)

The method of claim 1, wherein the tube has an internal surface, and wherein the internal surface of the tube is provided with hydrophobic and hydrophilic coating regions.

### Claim 11 (depends on 9)

The method of claim 9, wherein each droplet has a refractive index in the range 1.3 to 1.4 and the solvent has a refractive index in the range 1.3 to 1.7.

### Claim 12 (depends on 1)

The method of claim 1, wherein an array of tubes disposed in parallel in a substrate is employed and a stack of droplets is created in each tube.

### Claim 13 (depends on 12)

The method of claim 12, wherein the substrate is made of glass having a refractive index in the range 1.4 to 1.7 which is optionally light-absorbing.

### Claim 14 (depends on 1)

The method of claim 1, further comprising a step of irradiating the tubes with electromagnetic radiation and detecting the fluorescence radiation signal arising therefrom as each droplet emerges.

### Claim 15 (depends on 13)

The method of claim 13, wherein the refractive indices of the aqueous droplet, the solvent and glass are chosen so that guided modes are established which give rise to less than 10% cross-talk between the fluorescence signal generated by the emerging droplet and other droplets behind it in the stack.

### Claim 16 (depends on 14)

The method of claim 14, wherein scattering of electromagnetic radiation from one droplet being detected inhibits pick-up of electromagnetic radiation by the other droplets behind the one droplet in the stack by tuning wave-guided modes in the tube.

### Claim 17 (depends on 1)

A method as claimed in claim 1 suitable for characterising a biopolymer comprising the steps of (1) progressively digesting the biopolymer into an ordered stream of constituent monomers; (2) converting the stream of monomers into a corresponding stream of monomer-containing aqueous droplets, wherein each droplet additionally contains a probe capable of (a) capturing the monomer and (b) thereafter being digested to release an unquenched fluorophore characteristic of the captured monomer; (3) introducing the stream of droplets created in step (2) into an inlet end of at least one open-ended tube to create a stack of droplets therein; (4) releasing each droplet in turn from an outlet end of the tube(s); and (5) detecting fluorophores in each droplet as each droplet emerges.

### Claim 18 (depends on 17)

The method as claimed in claim 17, further comprising a step of (6) removing the droplet from the outlet prior to repeating steps (4) and (5) on the next emerging droplet.

### Claim 19 (depends on 17)

The method of claim 17, wherein the biopolymer is a nucleic acid and the monomers are nucleotides.

### Claim 20 (depends on 19)

The method of claim 19, wherein the nucleic acid is DNA or RNA of natural or synthetic origin.

### Claim 21 (depends on 17)

The method of claim 17, further comprising a period of incubation between steps (3) and (4).

### Claim 22

An apparatus for sequencing a biopolymer comprising:
a digestion unit including an analyte-receiving location where the biopolymer is progressively digested into a corresponding stream of constituent monomers;
a dispensing unit including at least one droplet-dispensing nozzle configured for dispensing the stream of monomers as a corresponding stream of droplets;
a substrate through which is provided an array of open-ended tubes each having an inlet and an outlet on opposing faces and whose major axes are disposed parallel to the direction in which the droplets are to be dispensed;
a means for stepping the droplet-dispensing nozzle(s) relative to the inlet face of the substrate in at least one direction perpendicular to the major axes of the tubes thereby allowing the droplets to be dispensed into the tubes;
a controller programmed to create a droplet stack in the tubes for releasing the droplets from the stack through the outlets as required;
at least one source of electromagnetic radiation configured to illuminate the outlets in the outlet face of the substrate;
a means for synchronising the illumination of the outlets as the droplets emerge therefrom, and
at least one photodetector configured to detect synchronised fluorescence radiation signals arising from the outlets.

### Claim 23 (depends on 22)

The apparatus of claim 22, further comprising a wave-guide associated with each tube for reducing or eliminating cross-talk fluorescence signals from secondary droplets in the droplet stack.

### Claim 24 (depends on 22)

The apparatus of claim 22 further comprising a chamber or fluidic junction located between the digestion unit and the dispensing unit for introducing a pyrophosphatase.

### Claim 25 (depends on 22)

The apparatus of claim 22, wherein the substrate is made of light absorbing glass having a refractive index in the range 1.45 to 1.65.

### Claim 26 (depends on 22)

The apparatus of claim 22, wherein the tubes are capillaries comprising walls with hydrophobic and hydrophilic coating regions.

### Claim 27 (depends on 22)

The apparatus of claim 22, wherein the controller includes a means for providing a negative pressure between the inlet and the outlet, and a micro-electro-mechanical gate which can be opened and closed to control release of each droplet from the droplet stack through the outlet.

### Claim 28 (depends on 22)

The apparatus of claim 22, wherein the tubes are vertically aligned and the controller releases each droplet from the bottom of the droplet stack.

### Claim 29 (depends on 13)

The method of claim 13, wherein the refractive index of the glass is chosen so that guided modes are established which give rise to less than 10% cross-talk between the fluorescence signal generated by the emerging droplet, and other droplets behind it in the stack.