# US 10,864,520 B2 · Fluidic devices with freeze-thaw valves with ice-nucleating agents and related methods of operation and analysis

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- Number: US 10,864,520 B2
- Document ID: US10864520B2
- Status: Granted
- Kind: B2
- Application number: 15/742,662
- Filed: 2016-07-22
- Published: 2018-07-19
- Granted: 2020-12-15
- Earliest claimed: 2015-07-22
- Assignees: The University of North Carolina at Chapel Hill
- Applicants: The University of North Carolina at Chapel Hill
- Inventors: John Michael Ramsey; William Hampton Henley; Joseph Carl Gaiteri
- Web page: https://patentgrep.com/patent/US10864520B2

## CPC

- B01L 7/50: {Cryostats}
- B01L 3/502738: {characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746)}
- C07K 14/195: from bacteria
- C07K 14/21: from Pseudomonadaceae (F)
- C07K 14/27: Erwinia (G)
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- F16K 99/0032: {using phase transition or influencing viscosity}
- F16K 99/0036: {operated by temperature variations}
- F16K 99/0044: {using thermo-electric means}
- G01N 1/42: Low-temperature sample treatment, e.g. cryofixation
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/1894: Cooling means; Cryo cooling
- B01L 2400/0677: phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- C12N 2310/16: Aptamers
- F16K 2099/0084: {Chemistry or biology, e.g. "lab-on-a-chip" technology}
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- F16K: VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING {(devices for emptying and evacuating the excess liquid in valves or conduits F16L55/07)}
- 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)

## Abstract

Embodiments of the invention provide fluidic devices such as, but not limited to, microfluidic chips, with one or more freeze thaw valves (FTVs) employing one or more ice-nucleating agents (INAs), that can reliably operate to freeze at relatively higher temperatures and/or at faster rates than conventional microfluidic devices with FTV systems.

## Claims

### Claim 1

A fluidic analysis device, comprising:
at least one fluid channel comprising at least one freeze thaw valve and at least one ice nucleating agent (INA), wherein the at least one INA is a carbon-containing INA, and wherein the at least one INA nucleates ice formation,
wherein the at least one INA comprisese an ice-like lattice structure.

### Claim 2 (depends on 1)

The fluidic analysis device of claim 1, wherein the at least one INA is extracted or derived from an organism.

### Claim 3 (depends on 1)

The fluidic analysis device of claim 1, further comprising first and second substrates attached together to define a microfluidic chip as the fluid analysis device with the at least one fluid transport channel and a plurality of spaced apart freeze thaw valves along the at least one fluid transport channel as the at least one freeze thaw valve.

### Claim 4 (depends on 1)

The fluidic analysis device of claim 1, in fluid communication with a liquid buffer comprising the at least one INA, wherein the at least one INA is flowably introduced into a fluid port on the fluidic analysis device, and wherein the at least one INA is present in the liquid buffer in an amount from 1 molecule/μL to 10 billion molecules/μL, or in a concentration of about 1 nM to about 1 mM.

### Claim 5 (depends on 1)

The fluidic analysis device of claim 1, wherein the at least one freeze thaw valve is a plurality of spaced apart freeze thaw valves, each comprising a thermoelectric cooler in thermal communication with a defined region of the at least one fluid channel, and wherein the at least one fluid channel has at least a segment that is sized to be a microfluidic or nanofluidic channel.

### Claim 6 (depends on 1)

The fluidic analysis device of claim 1, wherein the at least one fluid channel comprises a primary transport channel and at least one reagent channel, and wherein the fluidic analysis device comprises a sample input, a buffer input and a bead well array, all in fluid communication with the primary transport channel, with the at least one freeze thaw valve comprising a plurality of spaced apart freeze thaw valves in fluid communication with the primary transport channel and/or at least one reagent channel.

### Claim 7 (depends on 6)

The fluid analysis device of claim 6, further comprising a bead storage/sample incubation chamber, a waste reservoir and at least first and second freeze thaw valves that are spaced apart.

### Claim 8 (depends on 1)

The fluid analysis device of claim 1, wherein the at least one INA comprises a structure that aligns water molecules into an ice-like lattice.

### Claim 9

A fluidic analysis device, comprising:
at least one fluid channel comprising at least one freeze thaw valve and at least one ice nucleating agent (INA), wherein the at least one INA is a carbon-containing INA, and wherein the at least one INA comprises an ice-nucleating protein and/or a functional fragment thereof, an ice-nucleating nucleic acid, an ice-nucleating lipid, and/or an ice- nucleating carbohydrate.

### Claim 10 (depends on 2)

The fluidic analysis device of claim 2, wherein the at least one INA comprises the ice-nucleating protein (INP) and/or the functional fragment thereof.

### Claim 11 (depends on 10)

The fluidic analysis device of claim 10, wherein the INP and/or the functional fragment thereof is encoded by at least one gene selected from the group consisting of: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV, inaW, inaX, and inaZ, and wherein the gene is found in or obtained from an organism selected from the group consisting of: Pseudomonas syringae, Ps. fluorescens KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, Xanthomonas campestris, E. carotovora, Ps. antarctica, Ps. aeruginosa, Ps. putida, Ps. viridiflava, Pa. agglomerans, E. ananas, and Ps. borealis.

### Claim 12 (depends on 9)

The fluidic analysis device of claim 9, wherein the at least one fluid channel comprises at least one fluid channel with the at least one INA bonded and/or coated to a surface thereof.

### Claim 13

A method of analyzing a target analyte, comprising: providing a fluidic analysis device with at least one fluidic channel in fluid communication with at least one freeze thaw valve;
introducing at least one ice nucleating agent (INA) into the at least one fluidic channel, wherein the at least one INA contains a carbon atom, and wherein the at least one INA nucleates ice formation; and
electronically selectively cooling the at least one freeze thaw valve to freeze the at least one freeze thaw valve using the at least one INA, wherein the at least one INA comprises an ice-like lattice structure.

### Claim 14 (depends on 13)

The method of claim 13, wherein the at least one freeze thaw valve is a plurality of spaced apart freeze thaw valves, and wherein the method comprises setting coolers thermally communicating with the at least one fluidic channel to a temperature between −100 degrees C. and −1 degree C. to freeze the freeze thaw valves.

### Claim 15 (depends on 13)

The method of claim 13, wherein the fluidic device is a fluidic microchip with a thickness between 0.1 mm and 10 mm, wherein the at least one freeze thaw valve is a plurality of spaced apart freeze thaw valves, and wherein at least some of the freeze thaw valves have thermoelectric coolers set to between about −100 degrees C. and about −1 degree C. for a freeze operation that occurs in three minutes or less, and wherein the electronically selectively cooling is carried out a plurality of times for a respective sample.

### Claim 16 (depends on 13)

The method of claim 13, wherein the device is a fluidic microchip, wherein the method further comprises introducing the target analyte into the at least one fluidic channel, and wherein the target analyte is an organic compound or inorganic compound.

### Claim 17 (depends on 13)

The method of claim 13, wherein the at least one INA is extracted or derived from an organism.

### Claim 18 (depends on 13)

The method of claim 13, wherein the liquid is a buffer, and wherein the at least one INA is present in the buffer.

### Claim 19 (depends on 13)

The method of claim 13, wherein the at least one INA is introduced by releasing the at least one INA from a surface comprising a coating of the at least one INA and/or the at least one INA is adsorbed and/or chemically bonded to a surface of the at least one fluidic flow channel.

### Claim 20 (depends on 13)

The method of claim 13, wherein the at least one INA is immobilized on or coupled to a solid support.

### Claim 21 (depends on 13)

The method of claim 13, wherein the at least one INA comprises a structure that aligns water molecules into an ice-like lattice.

### Claim 22

A method of analyzing a target analyte, comprising:
providing a fluidic analysis device with at least one fluidic channel in fluid communication with at least one freeze thaw valve;
introducing at least one ice nucleating agent (INA) into the at least one fluidic channel, wherein the at least one INA contains a carbon atom; and
electronically selectively cooling the at least one freeze thaw valve to freeze the at least one freeze thaw valve using the at least one INA,
wherein the at least one INA comprises an ice-nucleating protein and/or a functional fragment thereof, an ice-nucleating nucleic acid, an ice-nucleating lipid, and/or an ice- nucleating carbohydrate.

### Claim 23 (depends on 22)

The method of claim 22, wherein the at least one INA comprises the ice-nucleating protein (INP) and/or one or more functional fragments thereof.

### Claim 24 (depends on 23)

The method of claim 23, wherein the INP and/or the one or more functional fragment(s) thereof is encoded by at least one gene selected from the group consisting of: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV, inaW, inaX, and inaZ, and wherein the gene is found in or obtained from an organism selected from the group consisting of: Pseudomonas syringae, Ps. fluorescens KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, Xanthomonas campestris, E. carotovora, Ps. antarctica, Ps. aeruginosa, Ps. putida, Ps. viridiflava, Pa. agglomerans, E. ananas, and Ps. borealis.

### Claim 25

A method of preparing a sample, comprising:
providing a fluidic analysis device comprising a sample metering loop and at least one fluidic channel, wherein the sample metering loop and the at least one fluidic channel are in communication with at least one freeze thaw valve;
introducing a sample into the sample metering loop, wherein the sample comprises at least one ice nucleating agent (INA) that contains a carbon atom, wherein the at least one INA nucleates ice formation, and wherein the at least one INA comprises an ice-nucleating protein and/or a functional fragment thereof, an ice-nucleating nucleic acid, an ice-nucleating lipid, and/or an ice-nucleating carbohydrate; and
electronically selectively cooling the sample metering loop using the at least one freeze thaw valve to freeze the at least one freeze thaw valve and/or at least a portion of the sample in the sample metering loop.

### Claim 26 (depends on 25)

The method of claim 25, wherein the at least one fluidic channel comprises a primary transport channel and at least one reagent channel, and wherein the fluidic analysis device comprises a sample input, a buffer input and a bead well array, all in fluidic communication with the primary transport channel, and wherein the at least one freeze thaw valve is a plurality of spaced apart freeze thaw valves in communication with the primary transport channel and/or at least one reagent channel.

### Claim 27 (depends on 25)

The method of claim 25, wherein the fluidic analysis device further comprises a bead storage/sample incubation chamber and a waste reservoir, wherein the sample metering loop resides between the bead storage/incubation chamber and the sample input.

### Claim 28 (depends on 25)

The method of claim 25, wherein the sample metering loop has a volumetric capacity of between about 1μL to about 1 mL and is in fluid communication with first and second freeze thaw valves on respective end portions of the sample metering loop.

### Claim 29 (depends on 25)

The method of claim 25, wherein the at least one INA comprises the ice-nucleating protein (INP) and/or one or more functional fragment(s) thereof.

### Claim 30 (depends on 29)

The method of claim 29, wherein the INP and/or the one or more functional fragment(s) thereof is encoded by at least one gene selected from the group consisting of: iceE, iceH, inaA, inaE, inaF, inaK, inaPb, inaQ, inaU, inaV, inaW, inaX, and inaZ, and wherein the gene is found in or obtained from an organism selected from the group consisting of: Pseudomonas syringae, Ps. fluorescens KUIN-1, Erwinia herbicola, E. uredovora, Pantoea ananatis, Xanthomonas campestris, E. carotovora, Ps. antarctica, Ps. aeruginosa, Ps. putida, Ps. viridiflava, Pa. agglomerans, E. ananas, and/or Ps. borealis.

### Claim 31

A method of preparing a sample, comprising:
providing a fluidic analysis device comprising a sample metering loop and at least one fluidic channel, wherein the sample metering loop and the at least one fluidic channel are in communication with at least one freeze thaw valve;
introducing a sample into the sample metering loop, wherein the sample comprises at least one ice nucleating agent (INA) that contains a carbon atom, wherein the at least one INA nucleates ice formation; and
electronically selectively cooling the sample metering loop using the at least one freeze thaw valve to freeze the at least one freeze thaw valve and/or at least a portion of the sample in the sample metering loop,
wherein the sample comprises a plurality of cells, and wherein electronically selectively cooling the sample metering loop using the at least one freeze thaw valve to freeze the at least one freeze thaw valve and/or at least a portion of the sample in the sample metering loop lyses at least a portion of the plurality of cells.