Methods for analyzing the presence and concentration of multiple analytes using a diffusion-based chemical sensor

• US 6,277,641 B1
• Filed: 11/17/1999
• Issued: 08/21/2001
• Est. Priority Date: 09/26/1997
• Status: Expired due to Term

• Current Assignee: Washington University In St Louis
• Original Assignee: University of Washington
• Inventors: Yager, Paul
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US09/441,572
• Time in Patent Office: 643 Days
• Field of Search: 436/52, 436/53, 436/172, 436/177, 436/180, 422/81, 422/82, 422/82.01, 422/82.08, 422/82.05
• US Class Current: 436/52
• CPC Class Codes: G01N 2030/8429 adding modificating material
• CPC Class Codes: G01N 30/0005 Field flow fractionation
• 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 436/2575 Volumetric liquid transfer

Abstract

The present invention provides a microfabricated sensor and a method capable of rapid simultaneous measurement of multiple analytes in a fluid sample. The sensor is inexpensive, disposable and portable, and requires only microliters of sample, a particular advantage with precious fluids such as blood. The sensor utilizes diffusion between layered laminar streams rather than side by side streams. This allows multiple side by side channels for simultaneous detection of multiple analytes. In the sensor, a sample stream and a carrier stream flow in layers, one on top of the other, and one or more reagents are introduced to the bottom of the carrier stream through either a fluid or a solid reagent inlet. The reagent contains reagent particles which, in the presence of the analyte, have a detectable change in a property. The analyte diffuses into the carrier stream where it interacts with reagent particles and is detected by optical, electrochemical or other devices.

Claims

1. A method of detecting the presence of first analyte particles in a sample stream, comprising the steps of:

conducting said sample stream into a laminar flow channel;

conducting a carrier stream into said laminar flow channel such that said sample stream and said carrier stream flow in layered laminar streams;

conducting a first reagent, containing first reagent particles, into said laminar flow channel such that said reagent comes in contact with said carrier stream;

allowing said first analyte particles to diffuse into said carrier stream; and

detecting the reaction of said first analyte particles with said first reagent particles.

2. The method of claim 1 further comprising the step of conducting a second reagent, containing second reagent particles, into said channel.

3. The method of claim 2 wherein said first and second reagents are conducted into said channel in parallel.

4. The method of claim 2 wherein said first and second reagents are conducted into said channel in series.

5. The method of claim 1 wherein said channel has a Reynolds number R<

1.

6. The method of claim 5 wherein said channel has a Reynolds number R<

0.1.

7. The method of claim 1 wherein said first reagent is a fluid reagent.

8. The method of claim 7 wherein said first reagent particles are immobilized on beads, said beads carried within said fluid reagent.

9. The method of claim 8 wherein said beads further comprise a second reagent immobilized thereon.

10. The method of claim 1 wherein said first reagent is a solid reagent.

11. The method of claim 10 wherein said solid reagent comprises a reagent pellet recessed in a cavity in said channel.

12. The method of claim 10 wherein said pellet comprises reagent particles in a soluble reagent carrier.

13. The method of claim 1 wherein said sample stream has a volume <

20 microliter.

14. The method of claim 1 wherein said sample stream also contains larger particles, further including the step of filtering said sample stream before conducting said sample stream into said flow channel.

15. The method of claim 14 wherein said step of filtering comprises the step of separating by diffusion said larger particles from said first analyte particles.

16. The method of claim 15 wherein said step of filtering further comprises the steps of:

establishing a laminar flow stream comprising an extraction stream adjacent to an unfiltered-sample stream;

wherein said step of separating comprises diffusion of said first analyte particles, but not said larger particles, from said unfiltered-sample stream to said extraction stream, whereby said unfiltered-sample stream becomes a residual-sample stream and said extraction stream becomes said sample stream; and

further comprising the step of separating said residual-sample stream from said sample stream.

17. The method of claim 1 wherein said reaction causes a change in an optical property of said first analyte or first reagent particles and wherein said step of detecting comprises an optical measurement.

18. The method of claim 17 wherein said step of detecting comprises an absorbance measurement.

19. The method of claim 18 wherein said absorbance measurement is carried out continuously along a portion of said laminar flow channel.

20. The method of claim 19 wherein said absorbance measurement comprises illuminating said channel with a light source and photographing said channel with a camera.

21. The method of claim 20 wherein said camera is a digital camera.

22. The method of claim 17 wherein said step of detecting comprises a fluorescence measurement.

23. The method of claim 17 wherein said step of detecting comprises a luminescence measurement.

24. The method of claim 1 wherein said reaction causes a change in an electrical property of said first analyte or first reagent particles and wherein said step of detecting comprises an electrical measurement.

25. The method of claim 24 wherein said electrical measurement is performed at a plurality of sequential positions in said channel.

26. The method of claim 1 further comprising a calibration performed before or after sample measurement, said calibration comprising the steps of:

conducting a calibration stream into said laminar flow channel, said calibration stream containing a known concentration of first analyte particles;

conducting a carrier stream into said laminar flow channel such that said calibration stream and said carrier stream flow in layered laminar streams;

conducting a first reagent, containing first reagent particles, into said laminar flow channel such that said reagent comes in contact with said carrier stream;

allowing said first analyte particles to diffuse into said carrier stream;

detecting the reaction of said first analyte particles with said first reagent particles; and

calibrating the sensitivity of the detection of said first analyte.