# US 8,783,466 B2 · Continuous biomolecule separation in a nanofilter

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- Number: US 8,783,466 B2
- Document ID: US8783466B2
- Status: Granted
- Kind: B2
- Application number: 13/005,840
- Filed: 2011-01-13
- Published: 2011-05-19
- Granted: 2014-07-22
- Earliest claimed: 2005-10-06
- Assignees: Massachusetts Institute of Technology
- Applicants: Jongyoon Han; Jianping Fu
- Inventors: Jongyoon Han; Jianping Fu
- Web page: https://patentgrep.com/patent/US8783466B2

## CPC

- B01D 61/027: {Nanofiltration}
- B01L 3/502746: {characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves}
- 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}
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0896: Nanoscaled
- B01L 2400/0415: electrical forces, e.g. electrokinetic
- B01L 2400/0487: fluid pressure, pneumatics
- B01L 2400/086: using baffles or other fixed flow obstructions
- G01N 2015/0288: {Sorting the particles}
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- 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

This invention provides a method and an apparatus for quickly continuously fractionating biomolecules, such as DNAs, proteins and carbohydrates by taking advantage of differential bidirectional transport of biomolecules with varying physico-chemical characteristics, for example size, charge, hydrophobicity, or combinations thereof, through periodic arrays of microfabricated nanofilters. The passage of biomolecules through the nanofilter is a function of both steric and electrostatic interactions between charged macromolecules and charged nanofilter walls, Continuous-flow separation through the devices of this invention are applicable for molecules varying in terms of any molecular properties (e.g., size, charge density or hydrophobicity) that can lead to differential transport across the nanofilters.

## Claims

### Claim 1

A biomolecular sorter comprising:
a) a substrate;
b) a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate;
c) a sample inlet to said sorter;
d) at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows; and
e) at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows;
wherein said obstacles are so arranged as to form gaps between said obstacles, with horizontal gaps being of a depth less than vertical gaps between said obstacles, the depth of the horizontal gaps is at most 10 nm, said at least a first conduit and said at least a second conduit provide said electrostatic force field or hydrodynamic force field to a sieving matrix by acting as electro-current injectors or fluidic flow injectors which enables separation of materials introduced by a sample provided, wherein a fabrication process of said matrix defines a specified geometrical shape of a cross-section of said rows, said gaps comprise a first set of regions that includes nanofilters and a second set of regions that includes microfluidic channels, said sample transits through said first set and second set of regions, said first set of regions provides steric hindrance of said sample through said transit.

### Claim 2 (depends on 1)

The sorter of claim 1, wherein said depth of said vertical gaps are from about 0.1-10 μm.

### Claim 3 (depends on 1)

The sorter of claim 1, wherein said obstacles in said rows are laterally shifted with respect to each row.

### Claim 4 (depends on 1)

The sorter of claim 1, wherein said gaps form channels for fluid conductance, when fluid is introduced in said sorter.

### Claim 5 (depends on 4)

The sorter of claim 4, comprising microfluidic channels in fluid communication with said channels.

### Claim 6 (depends on 5)

The sorter of claim 5, wherein said channels comprise sample loading ports.

### Claim 7 (depends on 5)

The sorter of claim 5, wherein said channels comprise sample collection ports.

### Claim 8 (depends on 5)

The sorter of claim 5, wherein said microfluidic channels are in fluid communication with a reservoir.

### Claim 9 (depends on 8)

The sorter of claim 8, wherein voltage is applied to said reservoir.

### Claim 10 (depends on 9)

The sorter of claim 9, wherein said applied voltage is less than 1000 V.

### Claim 11 (depends on 8)

The sorter of claim 8, wherein pressure is applied to said reservoir.

### Claim 12 (depends on 1)

The sorter of claim 1, wherein said electrostatic force field or hydrodynamic force field is applied in pulse-field operation mode.

### Claim 13 (depends on 1)

The sorter of claim 1, wherein said electrostatic force field or hydrodynamic force field is applied in continuous-field operation mode.

### Claim 14 (depends on 1)

The sorter of claim 1, wherein said substrate comprises silicon.

### Claim 15 (depends on 1)

A microchip comprising the sorter of claim 1.

### Claim 16

A method of sorting a fluid mixture comprising a plurality of biopolymers, which vary in terms of the physico-chemical characteristics of each of said plurality of biopolymers, said method comprising the steps of:
f) loading a fluid mixture comprising a plurality of biopolymers in a biomolecular sorter comprising:
i. a substrate;
ii. a plurality of obstacles arranged at regular intervals in a plurality of rows, columns or combinations thereof on a surface of said substrate, wherein said obstacles are so arranged as to form gaps between said obstacles, with horizontal gaps being of a depth less than vertical gaps between said obstacles, and said gaps form channels for fluid conductance, when fluid is introduced in said sorter, the depth of the horizontal gaps is at most 10 nm;
iii. a sample inlet to said sorter;
iv. microfluidic channels in fluid communication with said channels;
v. sample collection ports in fluid communication with said channels;
vi. at least a first conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said rows;
vii. at least a second conduit for applying an electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows; and
g) applying said electrostatic force field or hydrodynamic force field parallel in direction to said rows, and said electrostatic force field or hydrodynamic force field parallel in direction to said columns, and perpendicular in direction to said rows, whereby applying said force fields allows for separation of said plurality of biopolymers through said channels, said at least a first conduit and said at least a second conduit provide said electrostatic force field or hydrodynamic force field to a sieving matrix by acting as electro-current injectors or fluidic flow injectors which enables separation of materials introduced by said biopolymers, wherein a fabrication process of said matrix defines a specified geometrical shape of a cross-section of said rows, said gaps comprise a first set of regions that includes nanofilters and a second set of regions that includes microfluidic channels, said biopolymers transit through said first set and second set of regions, said first set of regions provides steric hindrance of said biopolymers through said transit; and
h) collecting separated biopolymers obtained in (b) from said sample collection ports.

### Claim 17 (depends on 16)

The method of claim 16, wherein said electrostatic force field parallel in direction to said rows provides an electroosmotic driving force for said fluid.

### Claim 18 (depends on 16)

The method of claim 16, wherein said fluid has an ionic strength of about 1-1000 mM.

### Claim 19 (depends on 18)

The method of claim 18, wherein said sorting is size-based.

### Claim 20 (depends on 16)

The method of claim 16, wherein said physico-chemical characteristics comprise size, charge, hydrophobicity, hydrophilicity, or a combination thereof.

### Claim 21 (depends on 16)

The method of claim 16, wherein said sorting is charge-based.

### Claim 22 (depends on 16)

The method of claim 16, wherein greater resolution of said biopolymers is achieved when said applied voltage is greater than 40 V.

### Claim 23 (depends on 16)

The method of claim 16, wherein said depth of said vertical gaps are from about 0.1-10 μm.

### Claim 24 (depends on 16)

The method of claim 16, wherein said obstacles in said rows are laterally shifted with respect to each row.

### Claim 25 (depends on 16)

The method of claim 16, wherein said sample inlet is a microchannel, in fluid communication with said channels.

### Claim 26 (depends on 16)

The method of claim 16, wherein said microfluidic channels are in fluid communication with a reservoir.

### Claim 27 (depends on 26)

The method of claim 26, wherein voltage is applied to said reservoir.

### Claim 28 (depends on 26)

The method of claim 26, wherein said applied voltage is less than 1000 V.

### Claim 29 (depends on 28)

The method of claim 28, wherein pressure is applied to said reservoir.

### Claim 30 (depends on 16)

The method of claim 16, wherein said electrostatic force field or hydrodynamic force field is applied in pulse-field operation mode.

### Claim 31 (depends on 16)

The method of claim 16, wherein said electrostatic force field or hydrodynamic force field is applied in continuous-field operation mode.

### Claim 32 (depends on 16)

The method of claim 16, wherein said fluid mixture comprises a cell lysate or tissue homogenate.

### Claim 33 (depends on 16)

The method of claim 16, wherein said fluid mixture comprises a large sample of deoxyribonucleic acids (DNA), proteins, or a combination thereof.

### Claim 34 (depends on 16)

The method of claim 16, wherein said fluid mixture comprises a buffered solution.

### Claim 35 (depends on 34)

The method of claim 34, further comprising the step of sorting a sample of said mixture two or more times, wherein the pH or ionic strength of said buffered solution is varied at the time of said sorting.