Recovery of rare cells using a microchannel apparatus with patterned posts

• US 20060160243A1
• Filed: 01/18/2005
• Published: 07/20/2006
• Est. Priority Date: 01/18/2005
• Status: Active Grant

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Tang, Zhongliang, Bhatt, Ram S., Tsinberg, Pavel
• Granted Patent: US 8,158,410 B2
• Time in Patent Office: Days
• US Class Current: 436/177
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2200/141 Preventing contamination, tampering
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0825 Test strips
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 2400/0457 passive flow or gravitation
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: G01N 1/34 Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/40 Concentrating samples
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 33/491 by separating the blood components G01N15/05 takes precedence
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

A microflow apparatus for separating or isolating cells from a bodily fluid or other liquid sample uses a flow path where straight-line flow is interrupted by a pattern of transverse posts. The posts are spaced across the width of a collection region in the flow path, extending between the upper and lower surfaces thereof; they have rectilinear surfaces, have arcuate cross-sections, and are randomly arranged so as to disrupt streamlined flow. Sequestering agents, such as Abs, are attached to all surfaces in the collection region via a hydrophilic coating, preferably a hydrogel containing isocyanate moieties or a PEG or polyglycine of substantial length, and are highly effective in capturing cells or other targeted biomolecules as a result of such streamlined flow disruption.

Claims

1. A microflow apparatus for separating biomolecules, such as cells, from a bodily fluid or other liquid, which apparatus comprises:

a body having a flow path defined therein through which a bodily fluid containing target biomolecules can be caused to flow, the flow path having inlet means, outlet means, and a microchannel arrangement extending between said inlet and outlet means, which microchannel arrangement includes a collection region with a plurality of transverse separator posts located in said region,

said posts being integral with a base surface of said microchannel and projecting therefrom, said posts being located in an irregular pattern extending laterally across said flow path in said collection region so as to interrupt straight-line flow and streamlined flow of liquid through said region, and said surfaces of said collection region including said posts carrying sequestering agents that will bind with target biomolecules,

whereby disruption of streamlined flow throughout said collection region as a result of said pattern of said posts creates effective capture of target biomolecules in said collection region.

2. The apparatus of claim 1 wherein said posts have at least about 3 different cross sectional sizes and said posts are aligned substantially perpendicular to the base surface of said microchannel.

3. The apparatus of claim 2 wherein said sequestering agents on said surfaces in said collection region are coupled thereto by hydrophilic linkers or a hydrogel coating so that they will bind effectively with ligands on the exterior surfaces of target cells in the bodily liquid.

4. The apparatus of claim 1 wherein said inlet means includes a well that will hold between about 50 and about 500 μ

l of liquid.

5. The apparatus according to claim 1 wherein said substrate is flat and has two major flat surfaces, wherein said flow path is defined in one of said surfaces, wherein a flat plate is affixed to said one surface to close said flow path in said collection region, and wherein free ends of said posts are affixed to said flat plate.

6. The apparatus according to claim 1, wherein said substrate and/or a flat plate which closes said collection region is optically transparent so that optical detection means positioned adjacent said collection region can monitor parameters of biomolecules captured within said region and provide an output signal corresponding to a monitored parameter.

7. The apparatus of claim 1 wherein said collection region has a hydrophilic coating on all surfaces threof, to which coating said sequestering agents are directly or indirectly bound.

8. The apparatus of claim 7 wherein said hydrophilic coating is at least about 1 micron thick and is an isocyanate-functional polymer containing residues of PEG, PPG or a copolymer thereof that are polymerized by urethane bonds, to which coating said sequestering agents are covalently bound.

9. The apparatus of claim 7 wherein said hydrophilic coating is at least about 1 micron thick and is a linear PEG or polyglycine having a molecular weight of at least about 3,000 daltons and a terminus to which said sequestering agent is covalently bound.

10. The apparatus of claim 1 wherein said posts occupy between about 20% and about 75% of the cross section of said region when taken in any plane perpendicular to the flow path therethrough and wherein the number of posts in said collection region is such that the posts occupy between about 15% and about 25% of the volume of the longitudinal length of the region wherein said posts are located.

11. The apparatus of claim 1 wherein said posts are arranged in a random pattern throughout said region and are spaced at least about 60 μ

m from the closest adjacent post.

12. A method for separating biomolecules, such as cells, from a bodily fluid or other liquid, which method comprises:

causing a bodily fluid containing target biomolecules to flow downstream along a flow path from an inlet to an outlet, which flow path comprises a channel arrangement that includes a first microchannel having a collection region and

separating target biomolecules from the flowing liquid by

(a) interrupting straight-line flow of the liquid through said collection region by blocking such flow with a plurality of separator posts located in said region, which posts are integral with a base surface of said microchannel and project therefrom, said posts extending transverse to said flow path and being located in an irregular pattern that extends laterally across said region and disrupts streamlined flow therethrough, and

(b) capturing target biomolecules in the flowing liquid on surfaces in said collection region by binding the target biomolecules thereto via sequestering agents carried on surfaces of said collection region, which capture is effective because of the disruption of streamlined flow throughout said collection region.

13. The method of claim 12 wherein a sample of said bodily liquid is placed into a well at said inlet and drawn through said channel by vacuum.

14. The method of claim 12 wherein said liquid is caused to flow through said collection region at between about 0.5 and 5 mm/sec.

15. The method according to claim 12 wherein a second group of target biomolecules are separated in a second collection region located downstream from the first collection region, which second region also contains a set of separator posts, which second region surfaces carry different sequestering agents than those in said first collection region.

16. The method according to claim 12, wherein said flow path is defined in a body which is optically transparent and wherein optical detection means positioned adjacent the flow path monitors parameters of biomolecules captured by separator posts within the flow path and provides an output signal corresponding to a monitored parameter.

17. The method of claim 12 wherein said target biomolecules are cells and said sequestering agents in said collection region are coupled thereto by hydrophilic linkers or a hydrogel coating so that they effectively bind with ligands on exterior surfaces of the target cells.

18. The method of claim 12 wherein said sequestering agents are connected to all surfaces in said collection region via a hydrogel layer of an isocyanate-functional polymer containing residues of PEG, PPG or a copolymer thereof that are polymerized by urethane bonds.

19. The method of claim 12 wherein said sequestering agents are connected to all surfaces in said collection region via a linear PEG or polyglycine having a molecular weight of at least about 3000 daltons and a terminus to which said sequestering agent is covalently bound.

20. The method according to claim 12 wherein, following said capture by said sequestering agents, said flow path is washed to remove nonspecifically bound biomolecules, and following said washing, said captured cells are released from said collection region and collected downstream for analysis.