BEAD READER

• US 20110136250A1
• Filed: 11/29/2010
• Published: 06/09/2011
• Est. Priority Date: 11/30/2009
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: RAN, Boaz, Sluszny, Chanan, Nimri, Shay
• Granted Patent: US 8,927,294 B2
• Time in Patent Office: Days
• US Class Current: 436/164
• CPC Class Codes: G01N 15/0612 Optical scan of the deposits G01N15/0625 takes precedence
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2035/1046 Levitated, suspended drops
• 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 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 21/78 producing a change of colour
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method of determining a normalized quantity of an analyte adhering to beads in a detection area of a bead-based assaying system, the method comprising:

• • a) causing a complex of the analyte to fluorescently or chemically emit a first light, or to release a dye;
  • b) measuring an integrated intensity of the first light emitted from the beads in the detection area, or a concentration of the dye released from the beads in the detection area, or both;
  • c) causing light to interact with the beads in the detection area, the interaction not depending on whether or how much analyte is adhering to the beads;
  • d) measuring a second light resulting from the interaction with the beads which does not depend on the analyte; and
  • e) determining the normalized quantity of analyte from the integrated intensity of the first light or concentration of the dye or both, and from the measured second light.

Claims

1. A method of determining a normalized quantity of an analyte adhering to beads in a detection area of a bead-based assaying system, the method comprising:

a) causing a complex of the analyte to fluorescently or chemically emit a first light, or to release a dye;

b) measuring an integrated intensity of the first light emitted from the beads in the detection area, or a concentration of the dye released from the beads in the detection area, or both;

c) causing light to interact with the beads in the detection area, the interaction not depending on whether or how much analyte is adhering to the beads;

d) measuring a second light resulting from the interaction with the beads which does not depend on the analyte; and

e) determining the normalized quantity of analyte from the integrated intensity of the first light or concentration of the dye or both, and from the measured second light.

2. A method according to claim 1, wherein determining the normalized quantity of analyte comprises:

a) determining a quantity of analyte adhering to the beads in the detection area, from the integrated intensity of the first light or concentration of the dye or both;

b) estimating a quantity of beads in the detection area from the measured second light; and

c) normalizing the quantity of analyte using the estimated quantity of beads.

3. A method according to claim 2, wherein measuring the second light comprises acquiring an image of the detection area, and estimating the quantity of beads comprises counting beads in the image using image processing software.

4. A method according to claim 1, wherein measuring the second light comprises measuring an integrated intensity of the second light from the detection area, and determining the normalized quantity of analyte comprises using the integrated intensity of the second light.

5. A method according to claim 2, wherein estimating the quantity of beads comprises estimating using only an integrated intensity of the second light from the detection area, or using only the integrated intensity of the second light from the detection area and one or more calibration parameters.

6. A method according to claim 5, wherein estimating the quantity of beads comprises estimating a quantity proportional to the integrated intensity of the second light from the detection area.

7. A method according to claim 1, wherein the interaction comprises reflection of the light from the beads.

8. A method according to claim 1, wherein the interaction comprises refraction of the light through the beads.

9. A method according to claim 1, wherein the interaction comprises exciting fluorescent emission from the beads with the light.

10. A method according to claim 9, wherein the complex is caused to emit the first light at different ranges of wavelengths than the second light.

11. A method according to claim 9, wherein the complex is caused to emit the first light fluorescently with different time dependence than the second light, by illuminating the beads with excitation light with a spectrum that varies with time.

12. A method according to claim 1, wherein the interaction comprises blocking or absorption of the light by the beads, and reflecting from or passing through areas between the beads, by the light.

13. A method according to claim 1, wherein measuring the integrated intensity of the first light, the second light, or both, comprises measuring the light after it has passed through a filter.

14. A method according to claim 1, wherein the complex is caused to emit the first light, and the beads are located in same places when measuring the first light and the second light.

19. A method according to claim 1, wherein the beads are magnetic, also comprising concentrating the beads in the detection area according to the method of claim 18.

15. A system for assaying an analyte adhering to beads, the system comprising:

a) a detection area where the beads with the analyte adhering to them are held;

b) a first detector which measures one or more of an integrated intensity of a fluorescent or chemical emission of light from the beads, or a catalyzed release of a dye from the beads, depending on an amount of analyte adhering to them;

c) a light source which produces light that interacts with the beads in the detection area, the interaction not depending on whether or how much analyte is adhering to the beads;

d) a second detector, the same as or different from the first detector, which measures a light received from the beads as a result of the interaction;

e) a controller which uses the measurements of the first and second detectors to determine a normalized quantity of analyte adhering to the beads.

16. A system according to claim 15, also comprising a first filter situated to preferentially admit the first light to the first detector when the first detector is measuring the first light.

17. A system according to claim 15, also comprising a second filter situated to preferentially admit the second light to the second detector when the second detector is measuring the second light.

18. A method of concentrating magnetic beads, used for assaying an analyte, in a detection area of a cell, the method comprising vibrating the cell while attracting the beads to the detection area using a magnetic field.

20. A method according to claim 18, wherein vibrating the cell comprises vibrating with a predominant frequency between 30 and 200 Hz.

21. A method according to claim 20, wherein the predominant frequency is between 60 and 100 Hz.

22. A method according to claim 18, wherein vibrating the cell comprises vibrating with a peak to peak amplitude of at least 0.5 mm.

23. A method according to claim 22, wherein the peak to peak amplitude is at least 1 mm.

24. A method according to claim 18, wherein vibrating the cell comprises vibrating so that, for horizontal components, vertical components or both, for at least most of the beads, the mass of the bead times maximum acceleration of the bead would be between 0.3 and 10 times the magnetic force on the bead, if the bead were located at the edge of the detection area.

25. A system for assaying an analyte adhering to beads, the system comprising:

a) a detection cell with a resting surface for the beads;

b) a magnet situated to attract the beads towards a detection area of the resting surface, when the beads are located on the resting surface;

c) a vibrator which vibrates the detection cell in a manner that facilitates concentration of the beads in the detection area when the magnet is attracting the beads toward the detection area;

d) a first detector which measures one or more of an integrated intensity of a fluorescent or chemical emission of light from the beads in the detection area, or a concentration of a dye released from the beads in the detection area, depending on an amount of analyte adhering to the beads; and

e) a controller which uses the measurements of the first detector to determine a quantity of analyte adhering to the beads.

26. A system according to claim 25, also comprising:

a) a light source which produces light that interacts with the beads in the detection area, the interaction not depending on whether or how much analyte is adhering to the beads; and

b) a second detector, the same as or different from the first detector, which measures a second light received from the beads as a result of the interaction;

wherein the controller uses the measurements of the first and second detectors to determine a normalized quantity of analyte adhering to the beads.

27. A system according to claim 25, wherein the vibrator is capable of vibrating the detection cell so that, for at least some of the beads, the mass of the bead, times the maximum acceleration of the detection cell, is greater than the static frictional force between the bead and the resting surface when the magnet is attracting the bead, at least for some part of the resting surface outside the detection area.