SAMPLE PROBES AND METHODS FOR SAMPLING INTRACELLULAR MATERIAL

• US 20140011226A1
• Filed: 07/03/2012
• Published: 01/09/2014
• Est. Priority Date: 07/03/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Sampas, Nicholas M., Bernick, Kristin Briana
• Granted Patent: US 9,255,245 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: B01L 3/021 Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
• CPC Class Codes: C12M 33/04 by injection or suction, e.g. using pipettes, syringes, needles pipettes in general B01L3/02
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01Q 60/38 Probes, their manufacture, or their related instrumentation, e.g. holders
• CPC Class Codes: G01Q 80/00 Applications, other than SPM, of scanning-probe techniques manufacture or treatment of nanostructures B82B3/00; recording or reproducing information using near-field interaction G11B9/12, G11B11/24, G11B13/08

Abstract

A sample probe includes a tip including a distal end for penetrating a cellular membrane, an opening located at or proximal to the distal end, and tip microchannels extending through the tip and communicating with the opening; and a body adjoining the tip and including body microchannels, wherein at least one of the body microchannels communicates with at least one of the tip microchannels. A method for sampling intracellular material includes inserting a probe tip through a cellular membrane; aspirating intracellular material from the cell, through an opening of the tip, and into a first microchannel of the tip; flowing isolator fluid from a second microchannel of the tip into the first microchannel to form a plug of intracellular material; and aspirating the plug and the isolator fluid through the first microchannel.

Claims

1. A sample probe, comprising:

a tip comprising a distal end configured for penetrating a cellular membrane, an opening located at or proximal to the distal end, and a plurality of tip microchannels extending through the tip and communicating with the opening; and

a body adjoining the tip and comprising a plurality of body microchannels, wherein at least one of the body microchannels communicates with at least one of the tip microchannels.

2. The sample probe of claim 1, wherein the body is a deflectable cantilever suitable for atomic force microscopy.

3. The sample probe of claim 1, wherein each tip microchannel has a cross-sectional dimension ranging from 0.1 μ

m to 100 μ

m.

4. The sample probe of claim 1, wherein the distal end comprises an annular apex circumscribing a cavity extending into the tip, and the opening communicates with the cavity.

5. The sample probe of claim 1, wherein the opening comprises a plurality of orifices, each orifice communicating with at least one of the tip microchannels.

6. The sample probe of claim 1, wherein one of the tip microchannels is a common microchannel interconnecting the opening with at least two other tip microchannels at a junction in the tip.

7. The sample probe of claim 1, wherein the plurality of tip microchannels or body microchannels comprises:

a first microchannel comprising a first section and a second section, the first section interconnecting the second section with the opening and having a first characteristic dimension, and the second section having a second characteristic dimension greater than the first characteristic dimension; and

a second microchannel communicating with the first microchannel at or near a junction between the first section and the second section.

8. The sample probe of claim 7, comprising a sensor communicating with the first microchannel and configured for producing a signal indicative of a type of fluid passing through the junction.

9. The sample probe of claim 1, comprising a first pump configured for transporting fluid from at least one of the tip microchannels to the opening, and a second pump configured for aspirating fluid from the opening to at least one other tip microchannel.

10. A method for sampling intracellular material, the method comprising:

inserting a tip of a probe through a membrane of a cell;

aspirating intracellular material from the cell, through an opening of the tip, and into a first microchannel of the tip;

flowing isolator fluid from a second microchannel of the tip into the first microchannel to form a plug of intracellular material defined in part by a boundary between the intracellular material and the isolator fluid; and

aspirating the plug and the isolator fluid through the first microchannel.

11. The method of claim 10, comprising repeating the steps of aspirating intracellular material and flowing isolator fluid into the first microchannel one or more times to form an alternating series of plugs of intracellular material and isolator fluid.

12. The method of claim 10, wherein the cell is a first cell and the plug of intracellular material is a first plug, and further comprising:

removing the tip from the first cell;

inserting the tip into a second cell;

aspirating intracellular material from the second cell, through the opening, and into the first microchannel; and

flowing isolator fluid from the second microchannel into the first microchannel to form a second plug of intracellular material from the second cell.

13. The method of claim 12, wherein the second plug is formed before aspirating the first plug to a downstream location, such that the second plug is isolated from the first plug in the first microchannel by the isolator fluid between the first plug and the second plug, and further comprising aspirating the first plug, the second plug and the isolator fluid to the downstream location in a single aspiration step.

14. The method of claim 10, wherein flowing the isolator fluid comprises:

flowing the isolator fluid from the second microchannel and through the opening to form a bulb of isolator fluid that blocks further aspiration of the intracellular material from the cell into the first microchannel; and

aspirating the isolator fluid through the opening and into the first microchannel.

15. The method of claim 10, wherein the tip comprises a third microchannel communicating with the opening and interconnecting the first microchannel and the second microchannel at a junction in the tip, and wherein the isolator fluid is flowed from the second microchannel into the first microchannel via the junction such that the isolator fluid at the junction blocks further aspiration of the intracellular material from the cell into the first microchannel.

16. The method of claim 10, comprising:

repeating the steps of aspirating intracellular material and flowing isolator fluid into the first microchannel one or more times to form an alternating series of plugs of intracellular material and isolator fluid; and

flowing the alternating series of plugs from the first microchannel into a downstream microchannel having a larger cross-sectional dimension than the first microchannel.

17. The method of claim 16, wherein the isolator fluid is immiscible with the intracellular material, and further comprising forming the plugs of the intracellular material into respective droplets in the downstream microchannel.

18. The method of claim 16, comprising maintaining contact between the plugs of the intracellular material and a wall defining the downstream microchannel while flowing the plugs of the intracellular material through the downstream microchannel.

19. The method of claim 16, comprising injecting an additional fluid into a flow of the alternating series of plugs at a location at or near an inlet of the downstream microchannel through which the plugs pass, wherein the additional fluid is selected from the group consisting of the isolator fluid, a buffer fluid, and both the isolator fluid and the buffer fluid.

20. A method for sampling intracellular material, the method comprising:

inserting a tip of a probe through a membrane of a cell, the tip comprising a first microchannel and a second microchannel;

injecting a buffer fluid from the second microchannel, through an opening of the tip, and into the cell, wherein the buffer fluid is miscible with the intracellular material and a portion of the intracellular material diffuses into the buffer fluid to form a zone of combined fluid in the cell;

aspirating the combined fluid through the opening and into the first microchannel;

intermittently flowing isolator fluid from a third microchannel into the first microchannel to form alternating plugs of combined fluid and isolator fluid, wherein the isolator fluid is immiscible with the combined fluid; and

aspirating the alternating plugs through the first microchannel.