# US 10,870,881 B2 · Biomolecular processing platform and uses thereof

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- Number: US 10,870,881 B2
- Document ID: US10870881B2
- Status: Granted
- Kind: B2
- Application number: 15/898,943
- Filed: 2018-02-19
- Published: 2018-07-05
- Granted: 2020-12-22
- Earliest claimed: 2013-02-08
- Assignees: Cornell University; The University of North Carolina at Chapel Hill; Northeastern University; Board of Supervisors of Louisiana State University and Agricultural and Mechanical College
- Applicants: Cornell University; Board of Supervisors of Louisiana State University and Agricultural and Mechanical College; University of North Carolina at Chapel Hill; Northeastern University
- Inventors: Steven A. Soper; Francis Barany; George Grills; Robin McCarley; Collin J. McKinney; Dorel Moldovan; Michael C. Murphy; Dimitris Nikitopoulos; Sunggook Park; Elizabeth J. Podlaha-Murphy
- Web page: https://patentgrep.com/patent/US10870881B2

## CPC

- C12Q 1/6869: Methods for sequencing
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- G01N 27/4473: {by electric means}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- G01N 33/48721: {Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68)}
- G01N 33/6803: {General methods of protein analysis not limited to specific proteins or families of proteins}
- H01J 49/009: {Spectrometers having multiple channels, parallel analysis}
- B01L 2200/027: for microfluidic devices
- B01L 2200/0663: Stretching or orienting elongated molecules or particles
- B01L 2300/0645: Electrodes
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0896: Nanoscaled
- B01L 2400/0415: electrical forces, e.g. electrokinetic
- B01L 2400/0421: electrophoretic flow
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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)
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)

## Abstract

The present invention relates to a device comprising a biomolecular processor. Each biomolecular processor has one or more bioreactor chambers defined by a solid substrate; a support structure within each bioreactor; a cleaving enzyme immobilized to the support structure and operatively positioned within the bioreactor chamber to cleave monomer or multimer units of a biopolymer molecule operatively engaged by the cleaving enzyme; and one or more time-of-flight channels formed in the solid substrate and fluidically coupled to said one or more bioreactor chambers. Each of the time-of-flight channels have two or more sensors including at least (i) a first sensor contacting the time-of-flight channel proximate to the input end of the channel and (ii) a second sensor contacting the time-of-flight channel proximate to the output end of channel. The present invention further relates to methods of sequencing and identifying biopolymer molecules using the device.

## Claims

### Claim 1

A device comprising:
a biomolecular processor, each biomolecular processor comprising:
one or more bioreactor chambers defined by a solid substrate;
a support structure within each bioreactor chamber and attached to the solid substrate;
a cleaving enzyme immobilized to the support structure and operatively positioned within said bioreactor chamber to cleave monomer or multimer units of a biopolymer molecule operatively engaged by said cleaving enzyme; and
one or more time-of-flight channels formed in the solid substrate and fluidically coupled to said one or more bioreactor chambers, each of said one or more time-of-flight channels having an input end and an output end, wherein each of said one or more time-of-flight channels comprise two or more sensors including at least (i) a first sensor contacting each of the one or more time-of-flight channels proximate to the input end of the time-of-flight channel and forming a first nanogap within the time-of-flight channel, wherein said nanogap has a width that is less than the width of the time-of-flight channel, and (ii) a second sensor contacting each of the one or more time-of-flight channels proximate to the output end of the time-of-flight channel and forming a second nanogap within the time-of-flight channel, wherein said second nanogap has a width that is less than the width of the time-of-flight channel.

### Claim 2 (depends on 1)

The device of claim 1 further comprising:
an electric field generator operatively positioned to create an electric field in said one or more bioreactor chambers and along the length of said one or more time-of-flight channels.

### Claim 3 (depends on 1)

The device of claim 1, wherein said one or more time-of-flight channels is less than or equal to 50 nm wide and less than or equal to 50 nm deep.

### Claim 4 (depends on 1)

The device of claim 1, wherein the first nanogap within the time-of-flight channel is about 1 nm to about 10 nm wide.

### Claim 5 (depends on 1)

The device of claim 1, wherein the second nanogap within the time-of-flight channel is about 1 nm to about 10 nm wide.

### Claim 6 (depends on 1)

The device of claim 1, wherein said one or more time-of-flight channels is 5 μm-250 μm long.

### Claim 7 (depends on 1)

The device of claim 1, wherein said one or more bioreactor chambers is 100 nm-1000 nm wide.

### Claim 8 (depends on 1)

The device of claim 1, wherein the support structure within the one or more bioreactor chambers is 50 nm-900 nm wide.

### Claim 9 (depends on 1)

The device of claim 1, wherein the solid substrate is a made of a polymeric material.

### Claim 10 (depends on 1)

The device of claim 1, wherein the cleaving enzyme is an exonuclease or exoribonuclease.

### Claim 11 (depends on 1)

The device of claim 1, wherein the cleaving enzyme is a protease.

### Claim 12 (depends on 1)

The device of claim 1, wherein the one or more time-of-flight channels comprise three or more sensors.

### Claim 13 (depends on 1)

The device of claim 1, wherein the biomolecular processor further comprises:
a feeder channel defined by walls of said solid substrate, said feeder channel having a length extending from an input end proximate to a surface of the solid substrate to an output end proximate to said one or more bioreactor chambers.

### Claim 14 (depends on 1)

The device of claim 1 further comprising:
a sensor contacting said feeder channel proximate to the input end of the feeder channel.

### Claim 15 (depends on 14)

The device of claim 14, wherein the feeder channel sensor forms a nanogap within the feeder channel, wherein said nanogap has a width that is less than the width of the feeder channel.

### Claim 16 (depends on 13)

The device of claim 13, wherein the feeder channel comprises one or more entropic traps between the input and output ends.

### Claim 17 (depends on 13)

The device of claim 13, wherein the feeder channel is less than or equal to 100 nm wide and less than or equal to 100 nm deep.

### Claim 18 (depends on 1)

The device of claim 1, wherein one or more the time-of-flight channels comprise a charge neutral hydrophilic wall surface, a charged hydrophilic wall surface, or a charge neutral hydrophobic wall surface.

### Claim 19 (depends on 1)

The device of claim 1, wherein the device comprises 10,000 or more biomolecular processors.

### Claim 20 (depends on 1)

The device of claim 1, wherein the device comprises 100,000 or more biomolecular processors.

### Claim 21 (depends on 1)

A method for nucleic acid molecule sequencing comprising:
providing the device of claim 1;
feeding a sample comprising one or more nucleic acid molecules into said biomolecular processor of said device under conditions effective for the immobilized cleaving enzyme within the bioreactor chamber to engage the one or more nucleic acid molecules in the sample and to cleave the one or more nucleic acid molecules into monomer nucleotides that individually enter the input end of the one or more time-of-flight channels;
applying an electric field across the one or more bioreactor chambers and along the length of the one or more time-of-flight channels to transport the cleaved monomer nucleotides through the one or more time-of-flight channels;
detecting, based on said applying, the cleaved monomer nucleotides as they pass at least the first and second sensors in the one or more time-of-flight channels;
measuring, based on said detecting, how long it takes for each cleaved monomer nucleotide to pass at least the first and second sensors in the one or more time-of-flight channels; and
identifying the monomer nucleotides based on said measuring.

### Claim 22 (depends on 21)

The method of claim 21, wherein said measuring further comprises:
measuring electrical peak amplitude of each cleaved monomer nucleotide as it passes at least one of the first or second sensors in the one or more time-of-flight channels.

### Claim 23 (depends on 1)

A method for identifying one or more proteins or polypeptides comprising:
providing the device of claim 1;
feeding a sample comprising one or more proteins or polypeptides into the biomolecular processor of the device under conditions effective for the immobilized cleaving enzyme within the bioreactor chamber to engage the one or more proteins or polypeptides in the sample and to cleave the one or more proteins or polypeptides into smaller peptide fragments that enter the input end of the one or more time-of-flight channels;
applying an electric field across the one or more bioreactor chambers and along the length of the one or more time-of-flight channels to transport the cleaved peptide fragments through the one or more time-of-flight channels;
detecting, based on said applying, the peptide fragments as they pass at least the first and second sensors in the one or more time-of-flight channels;
measuring, based on said detecting, (i) how long it takes for each peptide fragment to pass at least the first and second sensors of the one or more time-of-flight channels, and/or (ii) electrical peak amplitude of each peptide fragment as it passes at least one of the first or second sensors in the one or more time-of-flight channels; and
identifying at least a portion of the one or more proteins or polypeptides in the sample based on said measuring of the peptide fragments.