Scanning multifunctional particles

• US 9,476,101 B2
• Filed: 06/07/2011
• Issued: 10/25/2016
• Est. Priority Date: 06/07/2010
• Status: Active Grant

• Current Assignee: Firefly BioWorks, Inc. (Abcam Plc)
• Original Assignee: Firefly BioWorks, Inc. (Abcam Plc)
• Inventors: Pregibon, Daniel C., Marini, Davide M., Stoner, Isaac, Windemuth, Andreas, Erps, Timothy
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US13/702,860
• Publication Number:
  US 20130210653A1
• Time in Patent Office: 1,967 Days
• Field of Search: 435/6.1, 422/82.05
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2565/626 being a flow cytometer
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 21/47 Scattering, i.e. diffuse reflection G01N21/25, G01N21/41 take precedence G01N21/55 takes precedence
• 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 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The present invention provides, among other things, methods and systems for characterizing multifunctional objects using a flow-through device, such as, a flow cytometer. In some embodiments, an inventive method according to the present invention includes one more steps of (a) interrogating a plurality of objects (e.g., particles), wherein each individual object (e.g., particle) comprises one or more interrogation regions detectable as a sequence of events; (b) recording multiple events, wherein each individual event corresponds to each individual interrogation region detectable above a pre-determined triggering threshold; (c) grouping the recorded multiple events, and (d) characterizing the plurality of objects based on the grouped events.

Claims

1. A method for characterizing a multifunctional particle comprising:

(a) interrogating the multifunctional object particle with a flow cytometer, wherein the multifunctional particle comprises

(i) comprising at least one coding region and

(ii) at least one probe region,

wherein the at least one coding region and the at least one probe region are each doped with a triggering entity that is detectable above a pre-determined triggering threshold,

the at least one coding region and the at least one probe region being detectable by the flow cytometer as a sequence of discrete events above the pre-determined triggering threshold, and

wherein at least one non-detectable region separates the at least one coding region and the at least one probe region;

(b) detecting and recording, by the flow cytometer, the sequence of discrete events above the pre-determined triggering threshold, wherein each discrete event is designated by one or more numerical values indicative of a measured fluorescence and/or scatter signal; and

(c) characterizing the sequence of discrete events based on the numerical values thereby characterizing the coding and probing regions of the multifunctional particle.

2. The method of claim 1, wherein the sequence of discrete events are recorded non-contemporaneously.

3. The method of claim 1, wherein the sequence of discrete events are recorded contemporaneously.

4. The method of claim 1, wherein the multifunctional particle is illuminated with one or more lasers or light sources.

5. The method of claim 1, wherein the multifunctional particle comprises two coding regions and one probe region separated by inert spacers.

6. The method of claim 1, wherein the probe region contains DNA, RNA, proteins, and/or cells.

7. The method of claim 1, wherein the probe region comprises a generic linking chemistry.

8. The method of claim 1, wherein the probe region contains a single-stranded polynucleotide template containing capturing sequence specific for a target nucleic acid and an adjacent adapter sequence for binding a universal adapter such that a simultaneous binding of the target nucleic acid and the universal adapter is detectable via post-hybridization labeling.

9. The method of claim 1, wherein the multifunctional particle is a hydrogel particle.

10. The method of claim 1, wherein the at least one coding region comprises a first detectable label and the at least one probe region comprises a second detectable label, wherein the first detectable label and the second detectable label are distinct from each other.

11. The method of claim 10, wherein the first detectable label is a first fluorescent moiety and the second detectable label is a second fluorescent moiety.

12. A method for detecting multiple analytes in a sample comprising:

(a) mixing a plurality of multifunctional particles with a sample containing one or more target analytes, wherein each individual particle comprises

(i) at least one coding region and

(ii) at least one probe region,

wherein the at least one coding region and the at least one probe region are each doped with a triggering entity that is detectable above a pre-determined triggering threshold,

the at least one coding region and the at least one probe region being detectable by a flow cytometer as a sequence of discrete events above the pre-determined triggering threshold, and

wherein at least one non-detectable region separates the at least one coding region and the at least one probe region;

(b) detecting and recording, by the flow cytometer, the sequence of discrete events above the pre-determined triggering threshold, wherein each discrete event is designated by one or more numerical values indicative of a measured fluorescence and/or scatter signal;

(c) grouping the sequence of discrete events for each multifunctional particle;

(d) detecting the presence of the one or more target analytes by detecting altered events based on the grouping result from the step of grouping the events for each multifunctional particle as compared to a control.

13. The method of claim 12, wherein the sample is selected from cells, tissue, whole blood, plasma, serum, urine, stool, saliva, cord blood, chorionic villus samples amniotic fluid, transcervical lavage fluid, diseased cells (including cancer and/or tumor cells), and any combination thereof.

14. The method of claim 12, wherein the one or more target analytes comprise at least one of proteins, peptides, hormones, haptens, antigens, antibodies, receptors, enzymes, nucleic acids, polysaccharides, chemicals, polymers, pathogens, toxins, organic drugs, inorganic drugs, cells, tissues, microorganisms, viruses, bacteria, fungi, algae, parasites, allergens, pollutants, and any combination thereof.

15. The method of claim 14, wherein the one or more target analytes are nucleic acids comprising at least one of DNA, RNA, or any combination of DNA and RNA, a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, rRNA, microRNA, or any combination thereof.

16. The method of claim 12, wherein the one or more target analytes are present at a concentration ranging from 0.1 amol-10,000 amol in the sample.

17. The method of claim 12, wherein the one or more target analytes are present at a concentration below 100 amol in the sample.

18. The method of claim 12, wherein the one or more target analytes are present in a concentration of less than 1% of total nucleic acids in the sample.