Microfluidic device and surface decoration process for solid phase affinity binding assays

• US 20030124623A1
• Filed: 12/05/2002
• Published: 07/03/2003
• Est. Priority Date: 12/05/2001
• Status: Active Grant

• Current Assignee: Washington University In St Louis
• Original Assignee: Washington University In St Louis
• Inventors: Yager, Paul, Garcia, Elena
• Granted Patent: US 7,258,837 B2
• Time in Patent Office: Days
• US Class Current: 435/7.5
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/058 Flat flow cell
• CPC Class Codes: G01N 21/03 Cuvette constructions
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6456 : Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

This invention provides a microfluidic device for use in the detection of one or more analytes in a fluid using solid-phase affinity binding assays. The device offers a practical, easy-to-use, portable, inexpensive, robust analytical system for the parallel and quantitative detection of multiple analytes. In addition, this invention provides methods and devices for the formation of concentration gradients of capture molecules immobilized on a solid phase.

Claims

1. A microfluidic device for detecting the presence of an analyte in a fluid sample comprising:

a) a microfluidic channel comprising a bottom wall, two side walls and a top wall and having an upstream end and a downstream end;

b) a fluid inlet in fluidic connection with said microfluidic channel;

c) a storage area on one of said walls of said microfluidic channel downstream of said fluid inlet;

d) at least one solid reagent plug fixed in said storage area, said plug comprising a matrix material and a reagent having an affinity for binding to said analyte; and

e) a detection area within said microfluidic channel downstream of said fluid inlet and said storage area wherein said reagent binds to a binding wall of the microfluidic channel.

2. The microfluidic device of claim 1 wherein said detection area further comprises a linking reagent bound to said binding wall.

3. The microfluidic device of claim 1 wherein the microfluidic channel is made from a material selected from the group consisting of:

metals, polymers, glasses, and combinations thereof.

4. The microfluidic device of claim 1 wherein at least one of the walls in said detection area is transparent.

5. The microfluidic device of claim 1 wherein at least a portion of said binding wall is coated with a metal.

6. The microfluidic device of claim 5 wherein the wall opposite said binding wall also comprises a metal and wherein said two metal walls are capable of being in electrical connection with each other.

7. The microfluidic device of claim 1 wherein said storage area is a cavity positioned to allow said solid reagent therein to diffuse into a fluid flowing in said microfluidic channel.

8. The microfluidic device of claim 1 wherein said storage area is a spot upon which said solid reagent is fixed.

9. The microfluidic device of claim 1 further comprising a second storage area on said one of said walls of said microfluidic channel downstream from said inlet for introducing a second reagent into said laminar flow channel in contact with said carrier fluid stream.

10. The microfluidic device of claim 9 wherein said second storage area is positioned downstream from said first storage area.

11. The microfluidic device of claim 9 wherein said second storage area is positioned parallel to said first storage area.

12. The microfluidic device of claim 1 comprising a plurality of storage areas connected to said microfluidic channel downstream from said fluid inlet for introducing second and subsequent reagents into said microfluidic channel in contact with said fluid stream, said storage areas being positioned in parallel with each other.

13. The microfluidic device of claim 1 comprising a plurality of storage areas connected to said microfluidic channel downstream from said fluid inlet for introducing second and subsequent reagents into said microfluidic channel in contact with said fluid stream, said storage areas being positioned in series with each other.

14. The microfluidic device of claim 1 wherein said microfluidic device further comprises a plurality of storage areas connected to said microfluidic channel downstream from said fluid inlet for introducing second and subsequent reagents into said microfluidic channel in contact with said fluid stream, said storage areas being positioned in an array.

15. The microfluidic device of claim 1 wherein said microfluidic device further comprises:

a) a second inlet into said microfluidic channel in fluidic connection with a tributary microfluidic channel;

b) a second storage area disposed within said tributary microfluidic channel upstream of where said tributary channel joins said microfluidic channel; and

c) a second solid reagent plug fixed in said second storage area, said second reagent plug comprising a secondary reporter molecule having affinity for said analyte.

16. The microfluidic device of claim 1 wherein said solid reagent plug comprises a second reagent selected from the group consisting of:

a flow-rate monitoring compound, a dissolution-rate monitoring compound, and a pH indicator.

18. A method for storing a reagent in a microfluidic device comprising:

a) providing a microfluidic device;

b) forming a first reagent solution comprising said reagent and a matrix-forming material;

c) depositing said reagent solution in a storage area on said microfluidic device; and

d) converting said reagent solution in said storage area from a fluid to a solid to form a solid reagent plug and to form the microfluidic device of claim 1.

19. The method of claim 18 further comprising the step of monitoring said conversion.

20. A method for immobilizing a reagent in a microfluidic channel, comprising

a) providing a microfluidic device of claim 1;

b) flowing a fluid into said microfluidic channel through said inlet;

c) allowing said solid reagent plug to dissolve in said flowing solution whereby said dissolved or suspended reagent is carried to said detection area and immobilized on said binding wall.

21. The method of claim 20 wherein said reagent is a capture reagent.

22. The method of claim 20 whereby said immobilized reagent forms a concentration gradient on said binding wall.

23. The method claim 20 wherein said microfluidic device further comprises a linking reagent bound to said binding wall and said step of immobilizing comprises binding of said reagent to said bottom wall via said linking reagent.

24. The method of claim 20 wherein said reagent is biotinylated and said linking reagent is selected from the group consisting of avidin, streptavidin, and engineered molecules derived therefrom.

25. The method of claim 20 wherein said matrix material comprises a carbohydrate that is capable of replacing the waters of hydration of biomolecules in the absence of water.

26. The method of claim 20 wherein said matrix material is trehalose.

27. The method of claim 25 wherein said matrix material further comprises dextran.

28. A method of detecting an analyte in a sample using the microfluidic device of claim 1 comprising the steps of:

a) providing the microfluidic device of claim 1

b) flowing a first solution into said microfluidic device;

c) dissolving said solid reagent plug with said flowing solution, wherein said reagent is a capture reagent having affinity for said analyte;

d) carrying said dissolved reagent to said detection area;

e) immobilizing said reagent to said bottom wall within said detection area;

f) flowing a sample solution into said microfluidic device, said sample solution comprising the analyte to be detected;

g) binding said analyte to said capture reagent; and

h) detecting said bound analyte.

29. The method of claim 28 further comprising the step of determining the concentration of said analyte in said sample.

30. The method of claim 28 wherein said step of detecting comprises detecting using a method selected from the group consisting of:

optical absorption, fluorescence intensity, fluorescence intensity position, surface plasmon resonance, surface plasmon resonance microscopy, phosphorescence, chemiluminescence, electroluminescence, electrochemical, acoustic, and combinations thereof.

31. An analyte detection system comprising:

a) the microfluidic device of claim 1;

c) a fluid handling system in fluidic connection with a sample source and said inlet;

d) a detection system comprising a light source and a detector in optical connection with said detection area;

e) a computer coupled to said light source and said detector and optionally coupled to said fluid handling system.

32. The analyte detection system of claim 31 further comprising a device holder to secure said microfluidic device in optical connection with said detection area.

33. The analyte detection system of claim 31 in which said device holder further comprises a translation stage whereby said detection area is movable relative to said detection system.

34. The analyte detection system of claim 31 wherein at least a portion of said binding wall is coated with a metal and said optical detection system comprises:

a) a light source coupled to said binding wall through a prism whereby surface plasmon waves are excited at the metallic interior surface of said binding wall; and

b) a detector positioned to detect light reflected from the metallic surface of said binding wall through said prism

35. The analyte detection system of claim 31 wherein at least one wall of said detection area is transparent to light.

36. The analyte detection system of claim 35 wherein said optical detection system comprises:

a) a light source optically coupled to said detection area through said transparent wall; and

b) a detector optically coupled to said detection area through said transparent wall.

37. A method of making the microfluidic device of claim 1 comprising the steps of:

a) providing a first substrate layer;

c) providing a second layer in contact with said substrate layer wherein said second layer comprises microfluidic channel having an inlet, said bottom wall and said two side walls;

d) providing a third layer in contact with said second layer such that said third layer forms said top wall; and

e) immobilizing said solid reagent plug to one of said walls downstream of said fluid inlet to form said storage area.

38. The method of claim 37 comprising a further layer disposed between said substrate layer and said second layer, said further layer comprising a substrate with a cavity disposed thereon wherein said second layer further comprises a hole in alignment with said further layer.

17. A microfluidic device comprising:

a) a microfluidic channel comprising a bottom wall, two side walls and a top wall and having an upstream end and a downstream end;

b) a fluid inlet in fluidic connection with said microfluidic channel;

c) a storage area on one of said walls of said microfluidic channel downstream of said fluid inlet;

d) at least one solid reagent plug fixed in said storage area, said plug comprising a matrix material surrounding a reagent, wherein said reagent is selected from the group consisting of;

a flow-rate monitoring compound, a dissolution-rate monitoring compound, and a pH indicator.