Affinity binding-based system for detecting particulates in a fluid

• US 6,319,468 B1
• Filed: 07/12/2000
• Issued: 11/20/2001
• Est. Priority Date: 06/27/1995
• Status: Expired due to Fees

• Current Assignee: Tecan Trading AG (Tecan Group AG)
• Original Assignee: Tecan Trading AG (Tecan Group AG)
• Inventors: Sheppard, Norman F. Jr., Carvalho, Bruce L., Mian, Alec, Kellogg, Gregory, Kieffer-Higgins, Stephen G.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Do, Pensee T.
• Application Number:
  US09/614,834
• Time in Patent Office: 496 Days
• Field of Search: 422/63, 422/64, 422/67, 422/68.1, 422/50, 422/72, 422/101, 422/102, 436/45, 436/63, 436/177, 436/180, 210/787, 210/380.1, 210/374, 210/780, 210/109, 210/781, 435/7.1, 435/7.2, 435/7.21
• US Class Current: 422/63
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 35/71725 using centrifugal forces
• 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 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/024 Storing results with means integrated into the container
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0661 shape memory polymer valves
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• 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 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 7/00 : Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: C07K 14/79 : Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C12N 9/2462 : Lysozyme (3.2.1.17)
• CPC Class Codes: G01N 15/14 : Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 21/07 : Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/569 : for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
• CPC Class Codes: Y10S 366/03 : Micromixers: variable geometry from the pathway influences mixing/agitation of non-laminar fluid flow
• CPC Class Codes: Y10T 436/111666 : Utilizing a centrifuge or compartmented rotor
• 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

This invention provides methods and apparatus for detecting and quantifying particulate matter suspended in a fluid. Specifically, the invention provides an integrated, affinity-binding based, analytical system comprising a platform for performing an affinity-binding based assay for specifically binding particulates including microbial cells, and a detection device for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. Methods for using the analytical systems of the invention are also provided.

Claims

1. A centripetally-motivated apparatus for detecting a particulate in a fluid, the apparatus comprising in combination

a platform, comprising a substrate having a first flat, planar surface and a second flat, planar surface opposite thereto, and an axis of rotation, wherein the first surface defines a detection chamber comprising an area that is coated with a specific binding reagent that specifically binds to the particulate to be detected, and further comprises a multiplicity of microchannels embedded within the first planar surface and a sample input means, wherein the detection chamber, the sample input means and the microchannels are connected and in fluid communication, and wherein the platform further comprises

a wash buffer reservoir containing a wash buffer in fluid communication with the detection chamber;

and

a fluid waste receptacle in fluid communication with the detection chamber wherein the wash buffer reservoir and the sample input means are positioned on the first surface of the platform to be closer to the axis of rotation than is the detection chamber, and wherein the fluid waste receptacle is positioned on the surface of the platform to be farther from the axis of rotation than the detection chamber;

the apparatus comprising the platform in combination with

a device, comprising a base, a rotating means, a power supply and user interface and operations controlling means, wherein the rotating means is operatively linked to the platform and in rotational contact therewith

wherein a fluid sample comprising a particulate is moved from the sample input means to the detection chamber by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, and is incubated thereon for a time sufficient to result in specific binding between the particulate in the fluid sample and the specific binding reagent; and

wherein the fluid sample is replaced with the wash buffer and displaced into the fluid waste receptacle by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels; and

wherein the wash buffer is further displaced into the fluid waste receptacle by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels; and

wherein a particulate specifically bound to the detection chamber is detected thereupon.

2. An apparatus according to claim 1 wherein the surface of the detection chamber is a porous surface.

3. An apparatus of claim 1 wherein the sample input means further comprises a metering fluid sample application means, wherein a metered amount of an unmetered fluid sample applied to the sample input means is retained in the metering fluid sample application means, wherein the sample input means is further in fluidic contact with an overflow reservoir, wherein fluid comprising the unmetered fluid sample in excess of the metered amount of the fluid sample that is retained by the metering sample application means is transferred to the overflow reservoir.

4. An apparatus according to claim 3 wherein the metered amount of the fluid sample is from about 10 μ

L to about 500 μ

L.

5. The apparatus of claim 1, wherein the first flat, planar surface and second flat, planar surface of the platform form a disk.

6. The apparatus of claim 1, wherein the first and second flat, planar surfaces of the platform define a centrally located aperture that is engaged to a spindle on the device, whereby rotational motion of the spindle is translated into rotational motion of the platform.

7. An apparatus according to claim 1, wherein the particulate is a cell.

8. An apparatus according to claim 1, further comprising:

a reservoir comprising an amount of a detectable labeling moiety that is specific for the particulate to be detected and is in fluid communication with the detection chamber, wherein the wash buffer is displaced from the detection chamber by the detectable labeling moiety.

9. An apparatus according to claim 8, wherein the detectable labeling moiety is a histochemical stain.

10. An apparatus according to claim 9 wherein the detectable labeling moiety is a immunochemical reagent.

11. An apparatus according to claim 10 wherein the immunochemical reagent is a detectably labeled antibody.

12. An apparatus according to claim 11 wherein the detectable label is produced by an enzymatic moiety, wherein the apparatus further comprises a substrate for the enzymatic moiety wherein the substrate is converted to a detectable label by enzymatic action.

13. An apparatus according to claim 12 wherein the substrate comprises a component of the wash buffer.

14. An apparatus according to claim 12 wherein the substrate comprises a component of the reservoir containing the detectable labeling moiety.

15. An apparatus according to claim 8 wherein the detectable label is a fluorescent label.

16. An apparatus according to claim 1 wherein the wash buffer further comprises a detectable label.

17. An apparatus according to claim 1 wherein the detection chamber comprises a volume from about 5 μ

L to about 1000 μ

L.

18. An apparatus according to claim 1 wherein the device further comprises a light source positioned to illuminate the platform.

19. An apparatus according to claim 18 further comprising a photodetector, wherein the photodetector is positioned to detect light from the light source transmitted through or reflected from the detection chamber.

20. The apparatus of claim 19, wherein the photodetector is brought into alignment with the detection chamber by rotational motion of the platform.

21. The apparatus of claim 18, wherein the photodetector is stationary and samples the detection chamber at a frequency equal to the frequency of rotation of the platform or multiples thereof.

22. An apparatus according to claim 18 wherein a portion of the detection chamber is optically transparent, and the photodetector and the light source are positioned so that light from the light source illuminates the detection chamber and is detected by the photodetector through the optically transparent portion of the platform.

23. An apparatus according to claim 18 wherein a portion of the detection chamber comprises a reflective surface, and the photodetector and the light source are positioned so that light from the light source illuminates the detection chamber, is reflected therefrom and the reflected light is detected by the photodetector.

24. An apparatus according to claim 18 wherein the detection chamber further comprises alternating transparent and reflective regions.

25. An apparatus according to claim 24, wherein the alternating transparent and reflective regions define a pattern in the area.

26. An apparatus according to claim 24 wherein the specific binding reagent is present in the detection chamber on the surface of the platform on a transparent region thereof.

27. An apparatus according to claim 1 wherein the detection chamber is further treated with a blocking agent that prevents non-specific binding to the surface of the platform.

28. An apparatus according to claim 27 wherein the specific binding reagent is present in the detection chamber on the surface of the platform on transparent regions thereof, and the blocking reagent is present in the detection chamber on the surface of the platform on transparent and reflective regions thereof.

29. A method for detecting a cell in a fluid using an apparatus according to claim 1, the method comprising the steps of:

applying an amount of a sample to the fluid sample input means of the apparatus;

moving the fluid sample from the fluid sample input means to the detection chamber;

incubating the fluid sample in the detection chamber and replacing the fluid of the fluid sample in the detection chamber with the wash buffer, wherein the cells are retained in the detection chamber;

displacing the wash buffer into the fluid waste receptacle, wherein the cells are retained in the detection chamber; and

detecting the particulate retained in the detection chamber.