Chip for performing an electrophoretic separation of molecules and method using same

• US 6,254,754 B1
• Filed: 07/26/1999
• Issued: 07/03/2001
• Est. Priority Date: 07/29/1998
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ross, Gordon, Kaltenbach, Patrick
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US09/361,063
• Time in Patent Office: 708 Days
• Field of Search: 204/450, 204/451, 204/452, 204/453, 204/454, 204/455, 204/600, 204/601, 204/602, 204/603, 204/604, 204/605, 204/548, 204/644, 204/459
• US Class Current: 204/548
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027

Abstract

A chip for performing an electrophoretic separation of micromolecular or macromolecular structures in two dimensions comprises a base substrate comprising a main surface. A channel is formed in the main surface of said base substrate in a first direction, wherein a first voltage is applicable across the channel in order to allow a separation of micromolecular structures or macromolecular structures in the first direction on the basis of a first separation mechanism. In addition, a two-dimensional separation matrix is formed in the main surface of the base substrate, wherein the separation matrix comprises a shallow recess and an array of stand-alone posts arranged in the recess. The recess extends perpendicular from the channel in a second direction, wherein a second voltage is applicable across the separation matrix in order to allow a separation of the structures on the basis of a second separation mechanism.

Claims

1. A chip for performing an electrophoretic separation of molecular structures in two dimensions, comprising:

a base substrate comprising a main surface;

a channel formed in said main surface of said base substrate in a first direction, a first voltage being applicable across said channel in order to allow a first separation of said molecular structures in said first direction on the basis of a first separation mechanism;

a two-dimensional separation matrix formed in said main surface of said base substrate, said separation matrix comprising a shallow recess and an array of stand-alone posts arranged in said recess, said recess extending perpendicular from said channel in a second direction, wherein a second voltage is applicable across said separation matrix in order to allow a second separation of said molecular structures on the basis of the basis of a second separation mechanism; and

means for preventing penetration of the molecular structures in said second direction when said first voltage is applied across said channel by restricting an electrical field caused by said first voltage to said channel.

2. The chip of claim 1, wherein the posts are formed and arranged in said recess such that distances between posts decrease from the channel-side end of the separation matrix to the end of the separation matrix opposite thereto in the second direction.

3. The chip of claim 2, wherein the posts are formed and arranged in said recess such that the decrease of the distances is a linear decrease.

4. The chip of claim 1, wherein said preventing means is formed by multiple barriers arranged orthogonal to said channel at the channel-side end in said recess.

5. The chip of claim 1, wherein said preventing means comprises means for applying a third voltage to said chip such that field components of the electrical field caused by said first voltage which are orthogonal to said channel are substantially compensated.

6. The chip of claim 1, wherein the channel comprises an inlet connected to a buffer reservoir and further connected to a sample cavity and a waste sample cavity such that a sample can be injected into said channel by applying an electrical field between said sample cavity and said waste sample cavity.

7. The chip of claim 1, further comprising a cover substrate, a main surface of said cover substrate being connectable to the main surface of said base substrate such that a membrane is interposed between said main surfaces of the base substrate and the cover substrate, wherein said main surface of said cover substrate is provided with a chamber facing said separation matrix when said cover substrate is connected to said base substrate, reagents for a post separation detection analysis being loadable into said chamber.

8. The chip of claim 7, further comprising means for applying an electrical field between said base substrate and said cover substrate for allowing electro-blotting of separated molecules onto said membrane.

9. The chip of claim 1, wherein said first separation mechanism is isoelectric focusing.

10. The chip of claim 1, wherein said second separation mechanism is according to the molecular weight of the molecular structures.

11. A method for performing an electrophoretic separation of molecules making use of a chip according to claim 1, comprising the steps of:

introducing a pH gradient into said channel;

injecting a sample volume into said channel;

applying an electrical field across said channel by turning on said first voltage and, thus, performing a separation of said molecular structures on the basis of said first separation mechanism;

turning-off said first voltage;

incubating the separated molecules with sodium dodecyl sulphate;

applying an electrical field across said separation matrix by turning on said second voltage and, thus, performing a separation of said molecular structures on the basis of said second separation mechanism.