Apparatus and methods for integrated sample preparation, reaction and detection

• US 9,074,250 B2
• Filed: 02/05/2013
• Issued: 07/07/2015
• Est. Priority Date: 10/19/2005
• Status: Active Grant

• Current Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Original Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Inventors: Bird, Dylan Hilmer, Ching, Jesus, Johnson, Bruce A., Moravick, Keith E., Richardson, Bruce
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/759,557
• Publication Number:
  US 20130266948A1
• Time in Patent Office: 882 Days
• US Class Current: 1/1
• CPC Class Codes: B01F 2101/23 Mixing of laboratory samples e.g. in preparation of analysing or testing properties of materials
• CPC Class Codes: B01F 25/60 Pump mixers, i.e. mixing within a pump
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 35/712 for feeding fluids
• CPC Class Codes: B01F 35/715 Feeding the components in several steps, e.g. successive steps
• CPC Class Codes: B01F 35/7174 using pistons, plungers or syringes
• 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 2200/028 Modular arrangements
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• 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 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/042 Caps; Plugs
• CPC Class Codes: B01L 2300/044 pierceable, e.g. films, membranes
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• 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 2300/1805 : Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2400/0406 : capillary forces
• CPC Class Codes: B01L 2400/043 : magnetic forces
• CPC Class Codes: B01L 2400/0478 : pistons
• CPC Class Codes: B01L 2400/0481 : squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0622 : distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0633 : with moving parts
• CPC Class Codes: B01L 2400/0644 : rotary valves
• CPC Class Codes: B01L 2400/0683 : mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/502 : with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50851 : specially adapted for heating or cooling samples laboratory heating apparatus B01L7/00; incubators C12M
• CPC Class Codes: B01L 3/523 : with means for closing or opening
• CPC Class Codes: B01L 3/527 : for a plurality of reagents
• CPC Class Codes: B01L 7/00 : Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: G01N 1/38 : Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 2035/0436 : with pre-packaged reagents, i.e. test-packs
• CPC Class Codes: G01N 35/1079 : with means for piercing stoppers or septums
• CPC Class Codes: Y10T 436/118339 : with formation of a segmented stream
• 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 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction

Abstract

Cartridges for the isolation of a biological sample and downstream biological assays on the sample are provided, as are methods for using such cartridges. In one embodiment, a nucleic acid sample is isolated from a biological sample and the nucleic acid sample is amplified, for example by the polymerase chain reaction. The cartridges provided herein can also be used for the isolation of non-nucleic acid samples, for example proteins, and to perform downstream reactions on the proteins, for example, binding assays. Instruments for carrying out the downstream biological assays and for detecting the results of the assays are also provided.

Claims

1. A method comprising:

(a) providing a composition comprising nucleic acids bound to magnetic particles in a first chamber of a cassette comprising the first chamber and a reaction chamber;

(b) eluting the nucleic acids from the particles in the first chamber to obtain an eluted nucleic acid sample;

(c) magnetically attracting the magnetic particles to a cavity of a valve member movably disposed within a valve housing, wherein the valve housing is at least partially disposed between the first chamber and a second chamber and comprises a first opening in fluid communication with the first chamber and a second opening in fluid communication with the second chamber;

(d) transferring at least a portion of the eluted nucleic acid sample to the reaction chamber in fluid communication with the first chamber;

(e) amplifying one or more target nucleic acid sequences in the eluted nucleic acid sample by combining the eluted nucleic acid sample and PCR reagents in the reaction chamber under conditions suitable for PCR.

2. The method of claim 1, wherein the nucleic acids are eluted in an elution buffer comprising MgCl₂.

3. The method of claim 1, wherein one or more of the PCR reagents are provided in lyophilized form in the reaction chamber such that the PCR reagents are reconstituted upon transfer of the eluted nucleic acid sample to the reaction chamber.

4. The method of claim 1, further comprising heating the eluted nucleic acid sample to above 70°

C. prior to transferring the eluted nucleic acid sample to the reaction chamber.

5. The method of claim 1, further comprising heating the eluted nucleic acid sample to above 95°

C. prior to transferring the eluted nucleic acid sample to the reaction chamber.

6. The method of claim 1, wherein transferring the eluted nucleic acid sample to the reaction chamber comprises applying negative pressure to the first chamber.

7. The method of claim 1, further comprising monitoring the PCR in real time.

8. The method of claim 1, wherein the particles are magnetic silica particles.

9. The method of claim 1 further comprising removing the magnetic particles from the first chamber while retaining substantially all of the eluted nucleic acid sample in the first chamber by moving the valve member from a first position in which the cavity is in fluid communication with the first chamber to a second position in which the cavity is fluidically isolated from the first chamber.

10. A method comprising:

(a) providing a composition comprising nucleic acids bound to magnetic particles in a first chamber of a cassette comprising the first chamber and a reaction chamber;

(b) eluting the nucleic acids from the particles in the first chamber to obtain an eluted nucleic acid sample;

(c) magnetically attracting the magnetic particles to a cavity of a valve member movably disposed within a valve housing, wherein the valve housing is at least partially disposed between the first chamber and a second chamber and comprises a first opening in fluid communication with the first chamber and a second opening in fluid communication with the second chamber;

(d) transferring at least a portion of the eluted nucleic acid sample to the reaction chamber in fluid communication with the first chamber; and

(e) amplifying one or more target nucleic acid sequences in the eluted nucleic acid sample by combining the eluted nucleic acid sample and PCR reagents in the reaction chamber under conditions suitable for PCR,

wherein a PCR master mix containing PCR reagents and substantially no MgCl₂ is disposed in the reaction chamber, and the nucleic acids are eluted in an elution buffer comprising MgCl₂.

11. The method of claim 10, wherein one or more of the PCR reagents are provided in lyophilized form in the reaction chamber such that the PCR reagents are reconstituted upon transfer of the eluted nucleic acid sample to the reaction chamber.

12. The method of claim 10, further comprising heating the eluted nucleic acid sample to above 70°

C. prior to transferring the eluted nucleic acid sample to the reaction chamber.

13. The method of claim 10, further comprising heating the eluted nucleic acid sample to above 95°

C. prior to transferring the eluted nucleic acid sample to the reaction chamber.

14. The method of claim 10, wherein transferring the eluted nucleic acid sample to the reaction chamber comprises applying negative pressure to the first chamber.

15. The method of claim 10, further comprising monitoring the PCR in real time.

16. The method of claim 10 further comprising removing the magnetic particles from the first chamber while retaining substantially all of the eluted nucleic acid sample in the first chamber by moving the valve member from a first position in which the cavity is in fluid communication with the first chamber to a second position in which the cavity is fluidically isolated from the first chamber.