MULTIPLEXED FLOW ASSAY BASED ON ABSORPTION-ENCODED MICRO BEADS

• US 20150105295A1
• Filed: 12/18/2014
• Published: 04/16/2015
• Est. Priority Date: 09/26/2012
• Status: Active Grant

• Current Assignee: Xerox Corporation (Xerox Holdings Corp.)
• Original Assignee: Palo Alto Research Center, Inc. (Xerox Holdings Corp.)
• Inventors: Kiesel, Peter, Martini, Joerg, Recht, Michael I.
• Granted Patent: US 9,759,657 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• 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 2021/6441 with two or more labels
• 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 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label

Abstract

Analysis of a system and/or sample involves the use of absorption-encoded micro beads. Each type of micro bead is encoded with amounts of the k dyes in a proportional relationship that is different from proportional relationships of the k dyes of others of the n types of absorption-encoded micro beads. A system and/or a sample can be analyzed using information obtained from detecting the one or more types of absorption-encoded micro beads.

Claims

1. An analyzer comprising:

k light sources configured to selectively stimulate light emission from k dyes, each k dye having an absorption spectrum that is distinguishable from absorption spectra of others of the k dyes, the selective stimulation the k dyes including stimulating each k dye without substantially stimulating others of the k dyes, the k dyes encoding n types of absorption-encoded micro beads, each type of absorption-encoded micro bead encoded with amounts of the k dyes in a proportional relationship that is different from proportional relationships of the k dyes of others of the n types of absorption-encoded micro beads;

one or more photosensitive detectors configured to sense the light emission from the k dyes and to generate an output signal; and

a processor configured analyze the output signal to detect the n types of absorption-encoded micro beads based on the light emission from the k dyes and to analyze one or more properties of a system using information obtained from one or more types of absorption-encoded micro beads present in a sample.

2. The system of claim 1, wherein excitation light emitted by the k light sources is modulated, respectively, at k frequencies, v₁, v₂, . . . v_k;

the processor is configured to calculate a frequency spectrum of the output signal of the photosensitive detector and to detect the n types of the absorption-encoded micro beads using the frequency spectrum of the output signal.

3. A method comprising:

using n types of absorption-encoded micro beads, each type of absorption-encoded micro bead encoded with k dyes in a proportional relationship that is different from proportional relationships of the k dyes of others of the n types of absorption-encoded micro beads, each k dye having an absorption spectrum that is distinguishable from absorption spectra of others of the k dyes, the n types of absorption-encoded micro beads being functionalized with recognition elements configured to interact with n target analytes, respectively;

providing excitation light configured to stimulate light emission from the k dyes encoding the absorption-encoded micro beads present in a sample, wherein each of the k dyes is selectively stimulated without substantially stimulating light emission from others of the k dyes;

providing excitation light configured to stimulate light emission from a label dye, the light emission indicative of a presence of one or more analytes in the sample;

sensing for light emanating from the micro beads, the light emanating from each micro bead including one or more of light emission from the k dyes and light emission from the label dye;

detecting one or more types of absorption-encoded micro beads present in the sample based on the light emanating from the micro beads; and

detecting one or more target analytes present in the sample based on the light emanating from the micro beads.

4. The method of claim 3, wherein the n types of absorption-encoded micro beads are functionalized with n recognition elements, respectively, and detecting the one or more target analytes present in the sample comprises identifying multiple target analytes present in the sample.

5. The method of claim 3, wherein the label dye comprises one or more of:

a fluorophore-conjugated antibody configured to bind to a target analyte causing an increase in the light emanating from the micro beads when the target analyte is present in the sample;

a fluorophore-conjugated antibody configured to bind to a competitor molecule that is displaced from a recognition element of the micro beads by a target analyte causing a decrease in the light emanating from the micro beads when the target analyte is present in the sample;

a fluorophore-conjugated competitor molecule which is displaced from a recognition element of the micro beads by a target analyte causing a decrease in the light emanating from the micro beads when the target analyte is present in the sample;

a fluorophore conjugated to a recognition element of the micro beads, wherein a quencher-conjugated competitor molecule is displaced from a fluorophore-conjugated recognition element of the micro bead by binding to a target analyte, causing an increase in the light emanating from the micro beads when the target analyte is present in the sample;

6. The method of claim 3, wherein:

providing the excitation light configured to stimulate light emission from the k dyes comprises providing k types of excitation light intensity modulated, respectively, at k frequencies, v₁, v₂, . . . v_k;

sensing for the light emanating from the micro beads comprises sensing for light emission modulated at the k frequencies; and

detecting the one or more types of absorption-encoded micro beads present in the sample comprises detecting based on the modulated light emission from the k dyes.

7. The method of claim 3, wherein:

providing the excitation light configured to stimulate light emission from the label dye comprises providing excitation light modulated at a frequency, v;

sensing for the light emanating from the micro beads comprises sensing for light emission modulated at frequency v; and

detecting the one or more target analytes present in the sample comprises detecting based on the light emission modulated at the frequency v.

8. The method of claim 7, wherein:

providing excitation light configured to stimulate light emission from the k dyes and providing excitation light configured to stimulate light emission from the label dye comprises providing excitation light modulated at a frequency, v, for at least one k dye and the label dye; and

sensing for the light emanating from the micro beads comprises separating light emission from the at least one k dye and light emission from the label dye using an optical filter.

9. The method of claim 3, wherein:

detecting the one or more types of absorption-encoded micro beads present in the sample comprises detecting the one or more types of absorption-encoded micro beads using a first detector channel; and

detecting the one or more target analytes present in the sample comprises detecting the one or more analytes using a second detector channel.

10. The method of claim 9, wherein:

the absorption-encoded micro beads present in the sample flow through a flow channel;

the first detector channel comprises a first photosensitive detector configured to sense for the light emission from the k dyes; and

the second detector channel comprises a second photosensitive detector configured to sense for the light emission from the label dye, wherein the first photosensitive detector is spaced apart from the flow channel by a first distance and the second photosensitive detector is spaced apart from the flow channel by a second distance, and the first distance is greater than the second distance.

11. A method comprising:

using n types of absorption-encoded micro beads, each type of absorption-encoded micro bead encoded with an encoder dye, the n types of absorption-encoded micro beads being functionalized with n recognition elements, respectively, the n recognition elements configured to interact with n target analytes, respectively;

providing excitation light configured to stimulate light emission from the encoder dye encoding the absorption-encoded micro beads present in a sample;

providing excitation light configured to stimulate light emission from a label dye associated with one or more of the n recognition elements;

sensing for light emanating from the micro beads, the light emanating from each micro bead including one or more of light emission from the encoder dyes and light emission from the label dye;

detecting one or more types of absorption-encoded micro beads present in the sample based on the light emanating from the micro beads; and

detecting one or more target analytes present in the sample based on the light emanating from the micro beads.

12. A system comprising:

k light sources configured to stimulate light emission from each of k dyes, respectively, each k dye having an absorption spectrum that is distinguishable from absorption spectra of others of the k dyes, the distinguishable absorption spectra of the k dyes allowing selective stimulation of each k dye without substantially stimulating others of the k dyes, the k dyes encoding n types of absorption-encoded micro beads, each type of absorption-encoded micro bead encoded with amounts of the k dyes in a proportional relationship that is different from proportional relationships of the k dyes of others of the n types of absorption-encoded micro beads, the n types of absorption-encoded micro beads being functionalized with n recognition elements, respectively, the n recognition elements configured to interact with n target analytes, respectively;

one or more photosensitive detectors configured to sense the light emission from the k dyes; and

a processor configured to detect the n types of micro beads present in a sample based on the light emission from the k dyes and to detect the n target analytes.

13. The system of claim 12, further comprising:

a label dye configured to interact with the one or more analytes or to interact with at least some of the n the recognition elements;

an additional light source that emits excitation light configured to stimulate light emission from the label dye; and

wherein the one or more photosensitive detectors are configured to sense the light emission from the label dye and the processor is configured to detect the presence of one or more of the n target analytes based on the light emission from the label dye.

14. The system of claim 12, further comprising at least one spatial filter, the at least one spatial filter arranged between one of the absorption-encoded micro beads present in the sample and at least one of the k light sources and the photosensitive detectors.

15. A method of fabricating micro beads, comprising:

encoding n types of micro beads with k dyes, each k dye having an absorption spectrum that is distinguishable from absorption spectra of others of the k dyes, wherein after the encoding, each type of micro bead includes the k dyes in a proportional relationship that is different from proportional relationships of the k dyes of others of the n types of micro beads, the absorption spectra of the k dyes allowing selective stimulation of light emission from each of the k dyes without substantially stimulating light emission from others of the k dyes; and

functionalizing the n types of micro beads with n recognition elements, respectively, the n recognition elements configured to interact with n target analytes, respectively.

16. The method of claim 15, wherein the k dyes comprise one or more of:

DiA and Pacific Orange;

Pacific Blue and SYTOX Blue;

PerCP and APC; and

chlorophyll a and chlorophyll b.

17. The method of claim 15, wherein functionalizing the n-types of micro beads comprises creating an association between the n recognition elements and at least one additional dye.

18. The method of claim 15, wherein the at least one additional dye comprises one or more of Cy 5 and RP-E.

19. The method of claim 15, wherein 2≦

k≦

3 and 20≦

n≦

1000.