# US 9,518,299 B2 · Apparatus and methods for parallel processing of micro-volume liquid reactions

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- Number: US 9,518,299 B2
- Document ID: US9518299B2
- Status: Granted
- Kind: B2
- Application number: 14/558,501
- Filed: 2014-12-02
- Published: 2015-03-26
- Granted: 2016-12-13
- Earliest claimed: 2000-02-18
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Matthew T. O'Keefe; Pamela K. Foreman
- Web page: https://patentgrep.com/patent/US9518299B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- B01F 33/3021: {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}
- B01F 33/30351: {using hydrophilic/hydrophobic surfaces}
- B01F 35/712: {for feeding fluids}
- B01F 35/7172: {using capillary forces}
- B01L 3/5025: {for parallel transport of multiple samples}
- B01L 3/50857: {using arrays or bundles of open capillaries for holding samples}
- C12Q 1/6883: for diseases caused by alterations of genetic material
- H01J 49/0418: {for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates}
- B01F 33/30: Micromixers
- B01F 2215/0431: Numerical size values, e.g. diameter of a hole or conduit, area, volume, length, width, or ratios thereof
- B01J 2219/00286: Reactor vessels with top and bottom openings
- B01J 2219/00317: Microwell devices, i.e. having large numbers of wells
- B01J 2219/0072: Organic compounds
- B01J 2219/00722: Nucleotides
- B01J 2219/0074: Biological products
- B01L 3/5088: {confining liquids at a location by surface tension, e.g. virtual wells on plates, wires (B01L3/50857 takes precedence)}
- B01L 2200/0642: Filling fluids into wells by specific techniques
- B01L 2300/0819: Microarrays; Biochips
- B01L 2300/0829: Multi-well plates; Microtitration plates
- B01L 2300/087: Multiple sequential chambers
- B01L 2300/10: Means to control humidity and/or other gases
- B01L 2300/165: Specific details about hydrophobic, oleophobic surfaces
- B01L 2400/0688: surface tension valves, capillary stop, capillary break
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q 2600/172: Haplotypes
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B 60/14: for creating libraries
- Y02P 20/582: Recycling of unreacted starting or intermediate materials
- 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
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)
- 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)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

Disclosed herein are apparatuses and methods for conducting multiple simultaneous micro-volume chemical and biochemical reactions in an array format. In one embodiment, the format comprises an array of microholes in a substrate. Besides serving as an ordered array of sample chambers allowing the performance of multiple parallel reactions, the arrays can be used for reagent storage and transfer, library display, reagent synthesis, assembly of multiple identical reactions, dilution and desalting. Use of the arrays facilitates optical analysis of reactions, and allows optical analysis to be conducted in real time. Included within the invention are kits comprising a microhole apparatus and a reaction component of the method(s) to be carried out in the apparatus.

## Claims

### Claim 1

A method for simultaneously conducting a plurality of polynucleotide reactions, the method comprising:
introducing a plurality of liquid samples comprising a nucleic acid into a plurality of holes of a substrate having a first surface and a second surface and the plurality of holes extending through the substrate from the first surface to the second surface;
coating openings of the plurality of holes on the first and second surfaces of the substrate with a hydrophobic fluid; and
simultaneously conducting a plurality of polynucleotide reactions on the plurality in at least some of the plurality of holes.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The method of claim 1, further comprising sealing the plurality of holes with an evaporation barrier.

### Claim 4 (depends on 3)

The method of claim 3, wherein the evaporation barrier comprises a silicone rubber sheet.

### Claim 5 (depends on 3)

The method of claim 3, wherein the evaporation barrier comprises a polymeric or glass cover slide.

### Claim 6 (depends on 5)

The method of claim 5, wherein the cover slide is optically clear.

### Claim 7 (depends on 6)

The method of claim 6, further comprising optically monitoring progress of the reactions in the plurality of holes.

### Claim 8 (depends on 7)

The method of claim 7, wherein optically monitoring further comprises monitoring by fluorescence spectroscopy.

### Claim 9 (depends on 1)

The method of claim 1, wherein each hole or set of holes is circumscribed by a narrow raised feature.

### Claim 10 (depends on 1)

The method of claim 1, further comprising supplementing the reactions with one or more reagents during the course of the reactions.

### Claim 11 (depends on 1)

The method of claim 1, further comprising affixing a target nucleic acid to a wall of a hole by surface tension or desiccation.

### Claim 12 (depends on 1)

The method of claim 1, wherein the plurality of liquid samples is obtained from a single sample comprising the target nucleic acid sequence.

### Claim 13 (depends on 12)

The method of claim 12 wherein the single sample is a biological sample.

### Claim 14 (depends on 1)

The method of claim 1, wherein the substrate comprises a sequence-specific polynucleotide amplification reaction component reversibly affixed to a wall of a hole.

### Claim 15 (depends on 14)

The method of claim 14, wherein the sequence-specific polynucleotide amplification reaction component is dissolved when the liquid sample is introduced into the hole.

### Claim 16 (depends on 1)

The method of claim 1, wherein the liquid samples are terminally diluted to the extent that an average number of the holes contain exactly one copy of the target nucleic acid.

### Claim 17 (depends on 1)

The method of claim 1, wherein haplotype information for a target nucleic acid sequence is generated.

### Claim 18 (depends on 17)

The method of claim 17, wherein a sequence-specific probe is used to detect a genetic polymorphism.