Method and Apparatus Using Electric Field for Improved Biological Assays

• US 20090032401A1
• Filed: 07/10/2008
• Published: 02/05/2009
• Est. Priority Date: 07/13/2007
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Santiago, Juan G., Khurana, Tarun, Ronaghi, Mostafa
• Granted Patent: US 8,277,628 B2
• Time in Patent Office: Days
• US Class Current: 204/549
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00

Abstract

Disclosed are a method and apparatus that use an electric field for improved biological assays. The electric field is applied across a device having wells, which receive reactants, which carry a charge. The device thus uses a controllable voltage source between the first and second electrodes, which is controllable to provide a positive charge and a negative charge to a given electrode. By controlled use of the electric field charged species in a fluid in a fluid channel are directed into or out of the well by an electric field between the electrodes. The present method involves the transport of fluids, as in a microfluidic device, and the electric field-induced movement of reactive species according to various assay procedures, such as DNA sequencing, synthesis or the like.

Claims

1. A device for carrying out biological assays, having at least one fluid channel and a reaction area with an opening exposed to the fluid channel and a bottom of the reaction area offset from the fluid channel, the device being constructed for fluid flow in a direction transverse to reaction area openings, comprising:

(a) a first electrode adjacent to the bottom of the reaction areas;

(b) a second electrode adjacent to the opening of the reaction areas; and

(c) a controllable voltage source connected to the first and second electrodes which is controllable to provide both an alternating voltage and a DC bias voltage between the first electrode and the second electrode to create an electric field between the first electrode and the second electrode;

whereby charged species in a fluid in the fluid channel are directed into or out of the reaction area by the electric field between the electrodes.

2. The device of claim 1 further comprising a reaction sensor coupled to the reaction area for detecting reactions in the reaction area.

3. The device of claim 2 wherein the reaction sensor comprises a fiberoptic faceplate coupled to a transparent electrode.

4. The device of claim 2 wherein the reaction sensor comprises a CMOS photosensitive element.

5. The device of claim 1 further wherein the reaction area is a well that is sized to contain only one bead.

6. The device of claim 5 further comprising beads that are negatively charged.

7. The device of claim 6 wherein the negatively charged beads are polystyrene.

8. The device of claim 6 wherein the beads are magnetic and the device further comprises a magnet.

9. The device of claim 1 wherein the electrode adjacent to the bottom is a thin layer of ITO, less than about 150 nm thick.

10. The device of claim 1 wherein reaction areas are defined in an inert, solid polymer selected from the group consisting of photoresist and PDMS.

11. The device of claim 1 wherein the electrodes comprise a dielectric coating on surfaces exposed to liquid in the fluid channel.

12. The device of claim 11 wherein the dielectric coating is one or more of poly-p-xylylene or silicon oxide or silicon nitride.

13. The device of claim 1 wherein the electrodes are formed by a wire grid.

14. A device for directing charged particle movement in a liquid, wherein said particles are directed into a reaction area, comprising:

(a) a first electrode coated with a dielectric material on one side of the liquid in the reaction area;

(b) a second electrode coated with a dielectric material on an opposite side of the liquid in the reaction area;

(c) a fluid flow channel transverse to the reaction area; and

(d) connections for a signal generator which applies both an AC voltage and a DC bias voltage to the first electrode and the second electrode, whereby the electrodes are constructed and arranged to generate an electric field between them.

15. The device of claim 14 wherein the reaction area is large enough for only a single bead.

16. A method for moving a charged molecular species in a microfluidic device, said species moving into a reaction area from a fluid channel communicating with the reaction area, comprising the steps of:

(a) flowing the charged molecular species in the fluid channel in a flow direction;

(b) providing an electric field having a positive end and a negative end across the reaction area; and

(c) directing the charged molecular species into the reaction area by applying a charge to the electric field in the reaction area opposite to the charge on the molecular species.

17. The method of claim 16, wherein the reaction area contains a second molecular species, and wherein said method results in a reaction between the charged molecular species and the second molecular species.

18. The method of claim 16 further comprising the step of reversing the electric field at a frequency of at least 100 kHz.

19. The method of claim 16 wherein the frequency is a minimum of 10 kHz and a maximum of 10 MHz.

20. The method of claim 16 wherein the bias voltage is at least 1 volt.

21. The method of claim 16 wherein the electric field has a strength of at least about 5V/cm.

22. The method of claim 16 wherein the bias voltage is reversed to direct the molecular species out of the reaction area.

23. The method of claim 16 wherein the electric field is an electric displacement field.

24. A method of enriching for nucleic acid-coated beads in a microfluidic device, comprising the steps of:

(a) flowing naked and nucleic acid-coated beads in a fluid channel in a flow direction, wherein the fluid channel communicates with a reaction area;

(b) providing an electric field having a positive end and a negative end across the reaction area;

(c) directing the nucleic acid-coated beads into the reaction area by applying a charge to the electric field opposite to the charge on the DNA-coated beads; and

(d) collecting any beads that were not directed into the reaction area.

25. The method of claim 24, wherein the microfluidic device contains a plurality of reaction areas, and wherein each reaction area is a well sized to contain only one bead.