Simultaneous particle separation and chemical reaction

• US 6,221,677 B1
• Filed: 06/18/1999
• Issued: 04/24/2001
• Est. Priority Date: 09/26/1997
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: University of Washington
• Inventors: Weigl, Bernhard, Kenny, Margaret A., Yager, Paul, Wu, Caicai
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): PHAM, MINH QUAN K
• Application Number:
  US09/335,930
• Time in Patent Office: 676 Days
• Field of Search: 436/52, 436/53, 436/172, 436/177, 436/178, 436/188, 436/518, 422/81, 422/82, 422/82.05, 210/85, 210/94, 210/96.1, 210/511, 210/634, 210/739, 210/745, 210/746, 210/198.2, 210/243, 210/805
• US Class Current: 436/518
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• 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 436/255 including use of a solid sorbent, semipermeable membrane, or liquid extraction
• CPC Class Codes: Y10T 436/2575 Volumetric liquid transfer

Abstract

This invention provides a method and apparatus for detecting the presence of analyte particles in a sample fluid also comprising larger particles, particularly blood. It exploits diffusion to provide simultaneous filtering of the larger particles and reaction of the analyte particles. A sample stream and a reagent stream join on the upstream end of a laminar flow reaction channel and flow in adjacent laminar streams. The reagents can be in solution or immobilized on a bead. The analyte particles diffuse from the sample stream into the reagent stream, leaving behind the larger particles in the residual sample stream. In the reagent stream the analyte particles react with reagent particles and form product particles, thereby creating a product stream. At the downstream end of the reaction channel, the residual sample stream and the product stream are divided. The product particles are then detected, preferably optically, in the product stream. Prior to detection, the product stream can undergo further filtering or separation, or can join a second reagent stream to form secondary product particles. This apparatus and method can be used to implement competitive immunoassays or sandwich immunoassays using enzymatically or fluorescently labeled immunoreagents. The apparatus and method can also be used to synthesize products, in which case two reagent streams join in the laminar flow reaction channel.

Claims

1. A method for reacting small primary particles from a primary stream also comprising larger particles, comprising the steps of:

conducting said primary stream into a laminar flow reaction channel;

separately conducting a reagent stream comprising reagent particles into said reaction channel, such that said primary stream and said reagent stream flow in adjacent laminar streams;

allowing said primary particles to diffuse from said primary stream into said reagent stream, and to react with said reagent particles and form detectable product particles, thereby converting said reagent stream into a product stream and said primary stream into a residual primary stream;

conducting said residual primary stream out of said reaction channel;

separately conducting said product stream out of said reaction channel; and

detecting said product particles.

2. The method of claim 1 further comprising the step of analyzing said residual primary stream.

3. The method of claim 1 wherein said primary stream is blood, said small primary particles are native antigens, and said first reagent particles are first antibodies.

4. A method for reacting primary particles from a primary stream, comprising the steps of:

conducting said primary stream into a first laminar flow reaction channel;

separately conducting a first reagent stream comprising first reagent particles into said first laminar flow reaction channel, such that said primary stream and said first reagent stream flow in adjacent laminar streams;

allowing said primary particles to diffuse from said primary stream into said first reagent stream, and to react with said first reagent particles and form first product particles, thereby converting said first reagent stream into a first product stream and said primary stream into a residual primary stream;

thereafter conducting a first companion stream into said first laminar flow reaction channel such that said first product stream and said first companion stream flow in adjacent laminar streams thereby converting said first product stream into a diffused first product stream and said first companion stream into a diffused first companion stream;

conducting said residual primary stream out of said first reaction channel; and

separately conducting said first diffused product stream out of said first reaction channel.

5. The method of claim 4 further comprising allowing particles in said diffused first product stream and said diffused first companion stream to react with each other to form second product particles.

6. The method of claim 5 further comprising detecting said second product particles.

7. The method of claim 4 wherein said primary stream contains larger, non-diffusing particles in addition to said primary particles.

8. The method of claim 7 wherein said primary stream is blood, said primary particles are native antigens, and said first reagent particles are first antibodies.

9. The method of claim 4 wherein said step of detecting said first product particles comprises a method selected from the group consisting of optical, electrical, calorimetric and chemical detection.

10. The method of claim 4 wherein said step of detecting comprises absorbance, luminescence or fluorescence detection.

11. The method of claim 4 wherein said first reagent particles are immobilized on beads.

12. The method of claim 11 further comprising the step of detecting said first product particles using single particle detection.

13. The method of claim 4 further comprising the step of analyzing said residual primary stream.

14. A method for detecting antigens in whole blood comprising the steps of:

conducting a primary stream comprising whole blood containing first antigens into a laminar flow reaction channel;

separately conducting into said reaction channel a reagent stream comprising fluorescently-labeled second antigens identical to said first antigens, complexed with antibodies to said antigens, to form a complex, such that said primary stream and said reagent stream flow in adjacent laminar streams in said channel;

allowing first antigens from said blood to diffuse from said primary stream into said reagent stream, and to react with said complex to displace fluorescently-labeled second antigens therefrom, thereby converting said reagent stream into a product stream and said primary stream into a residual stream;

conducting said residual stream out of said reaction channel;

separately conducting said product stream out of said reaction channel; and

detecting displaced fluorescently-labeled second antigens in said product stream.

15. The method of claim 14 wherein said product stream is conducted through a convoluted detection channel.

16. The method of claim 14 wherein said displaced fluorescently-labeled second antigens are measured by fluorescence polarization.

17. A method for detecting antigens in whole blood comprising the steps of:

conducting a primary stream comprising whole blood containing first antigens into a laminar flow reaction channel;

separately conducting into said reaction channel a reagent stream comprising beads having attached thereto fluorescently-labeled second antigens, identical to said first antigens, complexed with antibodies to said antigens to form a complex, such that said primary stream and said reagent stream flow in adjacent laminar streams;

allowing first antigens from said blood to diffuse from said primary stream into said reagent stream and to react with said complex to displace fluorescently-labeled second antigens on said beads;

thereafter detecting fluorescently-labeled beads and/or fluorescently-labeled second antigens.

18. The method of claim 17 comprising conducting a stream comprising said beads through a flow cytometer and detecting said fluorescently-labeled beads therein.

19. The method of claim 17 where said reaction channel is convoluted.

20. The method of claim 17 wherein said streams in said reaction channel form a residual stream comprising whole blood from which small particles have diffused into said reagent stream, and a product stream comprising said beads with antigen-antibody complexes attached thereto, said method further comprising:

conducting said residual stream out of said reaction channel; and

separately conducting said product stream out of said reaction channel via a detection channel.

21. The method of claim 20 wherein said detection channel is convoluted.