Method for detecting analytes

• US 9,643,180 B2
• Filed: 09/24/2009
• Issued: 05/09/2017
• Est. Priority Date: 09/24/2008
• Status: Active Grant

• Current Assignee: First Light Diagnostics, Inc.
• Original Assignee: First Light Biosciences, Inc.
• Inventors: Abrams, Ezra, Gite, Sadanand, Shinefeld, Lisa, Straus, Don, Siek, Gordon, Yantz, Greg
• Primary Examiner(s): Brown, Mindy G
• Application Number:
  US13/120,516
• Publication Number:
  US 20120149007A1
• Time in Patent Office: 2,784 Days
• Field of Search: None
• CPC Class Codes: A61B 5/117 Identification of persons methods or arrangements for recognising patterns, e.g. fingerprints, G06F18/00, G06V40/00; identification of persons by analysing their voice or speech G10L17/00
• CPC Class Codes: A61B 5/150022 for capillary blood or interstitial fluid
• CPC Class Codes: A61B 5/150213 Venting means
• CPC Class Codes: A61B 5/150221 Valves
• CPC Class Codes: A61B 5/150274 Manufacture or production processes or steps for blood sampling devices
• CPC Class Codes: A61B 5/150305 Packages specially adapted for piercing devices or blood sampling devices kits for diabetes A61M5/003; packaging in general B65D
• CPC Class Codes: A61B 5/150343 Collection vessels for collecting blood samples from the skin surface, e.g. test tubes, cuvettes sampling from within body cavities A61B5/150015, A61B5/150045
• CPC Class Codes: A61B 5/150351 Caps, stoppers or lids for sealing or closing a blood collection vessel or container, e.g. a test-tube or syringe barrel caps for bottles or containers in general B65D41/00
• CPC Class Codes: A61B 5/150412 Pointed piercing elements, e.g. needles, lancets for piercing the skin blade-like piercing elements A61B5/150442
• CPC Class Codes: A61B 5/150503 Single-ended needles hollow piercing element tip design A61B5/150396, needle sets for catheters A61M25/06, needles for bringing media into the body A61M5/32
• CPC Class Codes: A61B 5/150786 Optical identification systems, e.g. bar codes, colour codes
• CPC Class Codes: A61B 5/151 Devices specially adapted for taking samples of capillary blood, e.g. by lancets , needles or blades
• CPC Class Codes: A61B 5/15113 Manually triggered, i.e. the triggering requires a deliberate action by the user such as pressing a drive button
• CPC Class Codes: A61B 5/15186 Devices loaded with a single lancet, i.e. a single lancet with or without a casing is loaded into a reusable drive device and then discarded after use; drive devices reloadable for multiple use
• CPC Class Codes: A61B 5/157 Devices characterised by integrated means for measuring characteristics of blood
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/022 : Transponder chips
• CPC Class Codes: B01L 2300/042 : Caps; Plugs
• CPC Class Codes: B01L 2300/043 : Hinged closures
• CPC Class Codes: B01L 2300/046 : Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/048 : enabling gas exchange, e.g. vents
• CPC Class Codes: B01L 2300/0654 : Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0681 : Filter
• CPC Class Codes: B01L 2300/0864 : comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0406 : capillary forces
• CPC Class Codes: B01L 2400/0409 : centrifugal forces
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/043 : magnetic forces
• CPC Class Codes: B01L 2400/0457 : passive flow or gravitation
• CPC Class Codes: B01L 2400/0472 : Diffusion
• CPC Class Codes: B01L 2400/0481 : squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0677 : phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 2400/0683 : mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/5029 : using swabs
• CPC Class Codes: B01L 3/5082 : Test tubes per se
• CPC Class Codes: B01L 7/00 : Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: B01L 9/52 : Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
• CPC Class Codes: G01N 2021/0325 : Cells for testing reactions, e.g. containing reagents
• CPC Class Codes: G01N 2021/0346 : Capillary cells; Microcells
• CPC Class Codes: G01N 2035/00356 : Holding samples at elevated temperature (incubation)
• CPC Class Codes: G01N 2035/0401 : Sample carriers, cuvettes or reaction vessels
• CPC Class Codes: G01N 2035/0429 : Sample carriers adapted for special purposes
• CPC Class Codes: G01N 21/03 : Cuvette constructions
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• 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 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• 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: G01N 35/02 : using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations G01N35/0098 and G01N35/0099 take precedence
• CPC Class Codes: G01N 35/025 : having a carousel or turntable for reaction cells or cuvettes

Abstract

The invention provides an improved method for sensitive and specific detection of target molecules, cells, or viruses. The inventive method uses large area imaging to detect individual labeled targets complexed with a target-specific selection moiety. The invention eliminates wash steps through the use of target-specific selection through one or more liquid layers that can contain optical dye and density agents. By eliminating washes the invention simplifies instrumentation engineering and minimizes user steps and costs. The invention uses sensitive image analysis to enumerate individual targets in a large area, is scalable, and can be deployed in systems ranging in complexity from manual to highly automated.

Claims

1. A method for determining whether one or more targets is present in a sample, said method comprising:

a) providing a vessel comprising a detection surface with a detection area having a shortest linear dimension of ≧

1 mm;

b) combining the sample with signaling moieties, selection moieties, and optionally a crosslinker, wherein either

(i) signaling moieties and selection moieties bind to one or more targets when present in the sample to form complexes of said one or more targets, said signaling moieties, and said selection moieties;

or

(ii) signaling moieties and selection moieties bind to each other, and one or more targets when present in the sample compete with said binding thereby reducing the formation of complexes of said signaling moieties and said selection moieties;

or

(iii) signaling moieties and selection moieties bind to the crosslinker, and one or more targets when present in the sample compete with said binding thereby reducing the formation of complexes of said signaling moieties, said selection moieties, and said crosslinker;

c) providing any complexes of (b)(i)-(b)(iii) in a liquid overlying layer that overlays an underlying cushion layer adjacent to the detection surface of the vessel, wherein the underlying cushion layer is denser than the overlying liquid layer and separates the overlying liquid layer from the detection surface and wherein said underlying cushion layer comprises a dye that interferes with the transmission of light to or from the signaling moieties;

d) applying a selection force to move said selection moieties, either unbound or in a complexes of (b)(i), (b)(ii), or (b)(iii), in said overlying layer through said underlying cushion layer to deposit said selection moieties within a detection zone corresponding to said detection area, wherein the selection force does not move said one or more targets not bound to selection moieties into the detection zone; and

e) simultaneously detecting individual complexes of (b)(i), (b)(ii), or (b)(iii) within the detection zone, at magnification of less than 5×

, to determine whether said one or more targets is present in said sample, wherein said method does not comprise a washing step and wherein said targets measure less than 50 microns in at least two orthogonal dimensions, and wherein, for (b)(ii) and (b)(iii), the determining whether said one or more targets is present in said sample is made relative to a reference.

2. The method of claim 1, wherein said signaling moieties comprise photonic signaling character.

3. The method of claim 1, wherein said signaling moieties or selection moieties are conjugated to a category-binding molecule.

4. The method of claim 3, wherein said category-binding molecule is an antibody, an antigen, a lectin, a carbohydrate, a protein, a nucleic acid molecule, a ligand, a receptor, or a small molecule.

5. The method of claim 1, wherein said signaling moieties specifically bind to said targets.

6. The method of claim 1, wherein said targets are cells, viruses, or molecules.

7. The method of claim 6, wherein said cells are bacterial cells.

8. The method of claim 1, wherein said bacterial cells are Staphylococcus aureus cells, E. coli cells, or Bacillus anthracis cells.

9. The method of claim 6, further comprising contacting said cells with a second signaling moiety that specifically binds to an internal component of said cell.

10. The method of claim 9, wherein said internal component is a nucleic acid molecule or a lipid.

11. The method of claim 9, further comprising, after step (d), photobleaching said second signaling moiety and detecting said signaling moiety from step (b) within said overlying layer or said underlying cushion layer.

12. The method of claim 1, wherein said targets are molecules secreted by microbial cells.

13. The method of claim 1, wherein said signaling moieties are fluorescent particles.

14. The method of claim 1, wherein said signaling moieties comprise a DNA stain.

15. The method of claim 1, wherein, prior to step (b), said sample is combined with microbiological growth medium followed by incubation for more than 1 hour.

16. The method of claim 15, wherein said microbiological growth medium comprises an antibiotic or microbial growth inhibitor.

17. The method of claim 1, wherein said signaling moieties or said selection moieties are in dried form in said vessel in step (a).

18. The method of claim 1, wherein said underlying cushion layer is in dried form in said vessel in step (a).

19. The method of claim 18, wherein in step (c) a composition comprising said targets hydrates said underlying cushion.

20. The method of claim 19, wherein in step (c) a composition comprising said targets hydrates said overlying layer and said underlying cushion layer.

21. The method of claim 1, wherein said overlying layer further comprises a dye that interferes with the transmission of light to or from the signaling moieties.

22. The method of claim 1, wherein said targets are selected from human thyroid stimulating hormone, C. albicans or an antigen of Bacillus anthracis.

23. The method of claim 22, wherein said antigen is selected from lethal factor (LF), protective antigen (PA), and poly-D-γ

-glutamic acid (PDGA) capsule polypeptide.

24. The method of claim 1, wherein said targets are methicillin-resistant S. aureus (MRSA) cells.

25. The method of claim 1, wherein said targets are present in, or obtained from, a biological sample.

26. The method of claim 25, wherein said biological sample is human whole blood, serum, plasma, mucus, urine, bile, synovial joint fluid, spinal fluid, amniotic fluid, sweat, nasal or respiratory fluid, vaginal secretions, ejaculate, wound exudates, sputum or bronchoalveloar lavage fluids, feces, bone, hair, dried cells, skin samples, fresh, frozen, or fixed pathology samples, or swabs taken from nasal, nasopharyngeal, throat, axillar, perineal, or rectal sites.

27. The method of claim 1, wherein said selection force is gravity, magnetism, electrical potential, centrifugal force, centripetal force, buoyant density, or pressure.

28. The method of claim 1, wherein said signaling moieties comprise fluorophores, chemiluminescent agents, bioluminescent agents, resonance light scattering particles, light absorption or chromogenic signaling agents, quantum dots, or up-converting phosphors.

29. The method of claim 1, wherein said selection moieties comprise magnetic particles, silica particles, or ferritin.

30. The method of claim 1, wherein the complexes are those of step (b)(ii) or (b)(iii);

and the detection of said target occurs by a reduction in the number of complexes of (b)(ii) or (b)(iii) formed in the presence of the target compared to the number of complexes formed in the absence of the target.

31. The method of claim 1, wherein said vessel is an imaging well having an imaging depth of greater than 2 mm.

32. The method of claim 1, wherein the targets are cells, and step (e) determines the differential growth of said cells in the presence and absence of a growth inhibitor.

33. The method of claim 1, wherein said complexes are those of (b)(i).