# US 10,371,622 B2 · Device for high throughput sperm sorting

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- Number: US 10,371,622 B2
- Document ID: US10371622B2
- Status: Granted
- Kind: B2
- Application number: 13/830,333
- Filed: 2013-03-14
- Published: 2014-09-18
- Granted: 2019-08-06
- Earliest claimed: 2013-03-14
- Assignees: Inguran, LLC
- Applicants: Inguran, LLC
- Inventors: Johnathan Charles Sharpe; Nemanya Sedoglavich; Erin Koksal; Kris Buchanan; Blair Morad
- Web page: https://patentgrep.com/patent/US10371622B2

## CPC

- G01N 15/1484: {microstructural devices}
- 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}
- B01L 3/502776: {specially adapted for focusing or laminating flows}
- C12N 5/061: {Sperm cells, spermatogonia}
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2200/0663: Stretching or orienting elongated molecules or particles
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2400/0487: fluid pressure, pneumatics
- B01L 2400/0622: distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
- B01L 2400/0633: with moving parts
- G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This disclosure relates to a device in the form of a microfluidic chip. In particular, various features are incorporated into the microfluidic chip for aligning and orienting sperm in flow channels, as well as for separating selected subpopulations of sperm.

## Claims

### Claim 1

A microfluidic chip comprising:
a substrate;
a flow channel formed in the substrate and configured for a flow direction comprising;
a sample inlet located at a first vertical position for injecting sample into the flow channel;
one or more sheath inlets that provide sheath fluid to the flow channel;
a fluid focusing region having:
a lateral fluid focusing chamber,
a first vertical fluid focusing channel in fluid communication with the flow channel that diverts sheath fluid from the lateral fluid focusing chamber and reintroduces the diverted sheath fluid at a first location downstream of the sample inlet, the first vertical fluid focusing channel having a horseshoe shape, and
a second vertical fluid focusing channel, having a horseshoe shape, in fluid communication with the flow channel that diverts sheath fluid from the lateral fluid focusing chamber and reintroduces the diverted sheath fluid at a second location, wherein one of the first vertical fluid focusing channel or the second vertical fluid focusing channel is in fluid communication with the flow channel from above the flow channel and the other is in fluid communication with the flow channel from below the flow channel, and wherein the flow channel is contacted by a vertical fluid focusing channel on only one side at the first location and downstream of the first location the flow channel is contacted on only the opposite side at the second location;
an inspection region at least partially downstream of the fluid focusing region; and
at least a first outlet in fluid communication with the flow channel.

### Claim 2 (depends on 1)

The microfluidic chip of claim 1, wherein the first vertical fluid focusing channel provides a first vertical influence at the first location and wherein the second vertical fluid focusing channel provides a second vertical influence at the second location in the opposite direction as the first vertical influence.

### Claim 3 (depends on 1)

The microfluidic chip of claim 1, wherein the fluid focusing region further comprises: ultrasonic transducers for producing pressure waves in the focusing region of each flow channel.

### Claim 4 (depends on 1)

The microfluidic chip of claim 1, wherein the fluid focusing region further comprises an array of ultrasonic transducers for producing a standing pressure wave along the flow channel.

### Claim 5 (depends on 1)

The microfluidic chip of claim 1, further comprising a planar channel geometry for orienting sperm.

### Claim 6 (depends on 1)

The microfluidic chip of claim 1, further comprising a nozzle geometry on the interior of the flow channel for orienting sperm.

### Claim 7 (depends on 1)

The microfluidic chip of claim 1, further comprising one or more of the following channel features for orienting sperm: a chevron, a gentle ramp, a decompression-compression zone, an abrupt ramp, or a step.

### Claim 8 (depends on 1)

The microfluidic chip of claim 1, wherein the at least a first outlet comprises a first outlet and a second outlet, and the microfluidic chip further comprises a diverting mechanism.

### Claim 9 (depends on 8)

The microfluidic chip of claim 8, wherein the diverting mechanism comprises a bubble valve or an array of ultrasonic transducers.

### Claim 10 (depends on 1)

The microfluidic chip of claim 1, wherein the flow channel has an associated reflective surface or refractive element that redirects a side fluorescence produced by sperm in the flow channel.

### Claim 11 (depends on 10)

The microfluidic chip of claim 10, wherein the associated reflective surface redirects a 90 degree side fluorescence into an optical path which is spatially separated from a first fluorescence and which is substantially parallel to the first fluorescence, so the 90 degree side fluorescence and the first fluorescence are collected by separate detectors.

### Claim 12 (depends on 11)

The microfluidic chip of claim 11, wherein the first fluorescence comprises a forward fluorescence.

### Claim 13 (depends on 11)

The microfluidic chip of claim 11, wherein the first fluorescence comprises a back fluorescence.

### Claim 14 (depends on 10)

The microfluidic chip of claim 10, wherein the reflective surface is formed as a surface on the substrate.

### Claim 15 (depends on 10)

The microfluidic chip of claim 10, wherein the reflective surface is formed as a surface of the flow channel.

### Claim 16 (depends on 1)

The microfluidic chip of claim 1, wherein the one or more sheath inlets are in fluid communication with the a sheath source, and wherein the sample inlet is positioned within a sheath flow created by the one sheath inlet to facilitate a co-axial flow of sheath and sample.

### Claim 17 (depends on 16)

The microfluidic chip of claim 16, wherein the flow channel comprises a first width and a first height at the sample inlet.

### Claim 18 (depends on 17)

The microfluidic chip of claim 17, wherein the flow channel comprises a second width and a second height at a first transition point.

### Claim 19 (depends on 18)

The microfluidic chip of claim 18, wherein the width of the flow channel is reduced between the sample inlet and the first transition point.

### Claim 20 (depends on 18)

The microfluidic chip of claim 18, wherein the flow channel comprises a third width and a third height at a second transition point.

### Claim 21 (depends on 20)

The microfluidic chip of claim 20, wherein the width remains constant between the first transition point and the second transition point and the height is reduced between the first transition point and the second transition point.

### Claim 22 (depends on 21)

The microfluidic chip of claim 21 wherein the third height and the third width are maintained through the inspection region.

### Claim 23 (depends on 21)

The microfluidic chip of claim 21, wherein the fluid flow channel transitions from a square cross section to a non-square rectangular cross section.

### Claim 24 (depends on 21)

The microfluidic chip of claim 21, wherein the flow channel transitions from a circular cross section to a non-circular, elliptical cross section.

### Claim 25 (depends on 1)

The microfluidic chip of claim 1, wherein the first vertical fluid focusing channel contacts the flow channel at a first location and the second vertical fluid focusing channel contacts the flow channel at a second different location along the flow path.

### Claim 26 (depends on 25)

The microfluidic chip of claim 25, wherein there is no overlap between the first location and the second locations on the flow channel.

### Claim 27 (depends on 1)

The microfluidic chip of claim 1, wherein the flow channel comprises a channel geometry having a transition to a reduced flow channel height.

### Claim 28 (depends on 1)

The microfluidic chip of claim 1, the first vertical fluid focusing channel and the second vertical fluid focusing channel are arranged with the flow channel to produce a ribbon core stream, when in use.

### Claim 29 (depends on 1)

The microfluidic chip of claim 1, wherein the first vertical fluid focusing channel and the second vertical fluid focusing channel comprise a double horseshoe configuration for diverting sheath fluid from the flow channel to the first location and the second location.

### Claim 30 (depends on 1)

The microfluidic chip of claim 1, further comprising multiple flow channels formed on the substrate.