Universal sample preparation system and use in an integrated analysis system

• US 8,562,918 B2
• Filed: 12/17/2012
• Issued: 10/22/2013
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Jovanovich, Stevan B., Nielsen, William D., Cohen, David S., Recknor, Michael, Vangbo, Mattias, Van Gelder, Ezra, Majlof, Lars, El-Sissi, Omar
• Primary Examiner(s): Sines, Brian J
• Application Number:
  US13/717,585
• Publication Number:
  US 20130224846A1
• Time in Patent Office: 309 Days
• Field of Search: 422/50, 422/68.1, 422/81, 422/502, 422/503, 436/43, 436/63, 436/94, 436/180, 436/174
• US Class Current: 422/502
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• 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 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0672 Integrated piercing tool
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• 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 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/527 for a plurality of reagents
• 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: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01N 1/31 Apparatus therefor
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 2035/00148 : Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 2035/00247 : Microvalves
• CPC Class Codes: G01N 21/64 : Fluorescence; Phosphorescence
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 2201/062 : LED's
• CPC Class Codes: G01N 27/44704 : Details; Accessories
• CPC Class Codes: G01N 27/44721 : by optical means
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 27/745 : for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 35/00029 : provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The invention provides a system that can process a raw biological sample, perform a biochemical reaction and provide an analysis readout. For example, the system can extract DNA from a swab, amplify STR loci from the DNA, and analyze the amplified loci and STR markers in the sample. The system integrates these functions by using microfluidic components to connect what can be macrofluidic functions. In one embodiment the system includes a sample purification module, a reaction module, a post-reaction clean-up module, a capillary electrophoresis module and a computer. In certain embodiments, the system includes a disposable cartridge for performing analyte capture. The cartridge can comprise a fluidic manifold having macrofluidic chambers mated with microfluidic chips that route the liquids between chambers. The system fits within an enclosure of no more than 10 ft³. and can be a closed, portable, and/or a battery operated system. The system can be used to go from raw sample to analysis in less than 4 hours.

Claims

1. A system configured to perform a method comprising:

extracting DNA from a sample;

isolating the extracted DNA;

amplifying a plurality of short tandem repeat (STR) markers of the isolated DNA;

separating a plurality of amplified STR markers by electrophoresis;

detecting a plurality of separated STR markers; and

performing computer analysis of a plurality of detected STR markers to produce a computer file identifying a plurality of detected STR markers;

wherein the method is performed in less than 4 hours; and

wherein the system comprises;

a) a sample preparation module adapted to extract DNA from a sample in a non-microfluidic volume, to isolate the extracted DNA by capture of the extracted DNA to particles, and to move the particles comprising isolated DNA through a first microfluidic channel;

b) a reaction module comprising a reaction chamber in fluidic communication with the first microfluidic channel and adapted to immobilize the particles comprising isolated DNA and to perform an amplification reaction on the isolated DNA to produce a reaction product;

c) a separation and detection module in fluidic communication with the reaction chamber and adapted to separate the reaction product by electrophoresis and to detect the separated reaction product; and

d) a data analysis module adapted to receive data on the separation and detection of the reaction product from the separation and detection module and comprising computer-executable code that transforms the data and produces a computer file identifying the separated and detected reaction product;

wherein the sample preparation module and the reaction module are integrated in a disposable cartridge comprising;

i) at least one set of fluidic chambers comprising a sample chamber, a capture chamber and a reaction chamber adapted for thermal cycling, wherein the chambers are in fluidic communication with each other; and

ii) a reagent cartridge comprising reagents for performing an amplification reaction involving thermal cycling, wherein the reagent cartridge is configured to be carried on the disposable cartridge in a closed configuration and to be placed into fluidic communication with the at least one set of fluidic chambers.

2. The system of claim 1, wherein the method further comprises washing the isolated DNA to purify the isolated DNA prior to amplifying.

3. The system of claim 1, wherein the particles are magnetically responsive particles, the capture chamber comprises the particles, and the reaction module further comprises a source of magnetic force configured to immobilize the particles comprising isolated DNA.

4. The system of claim 1, wherein the reagents in the reagent cartridge comprise reagents for performing PCR.

5. The system of claim 1, wherein the at least one set of fluidic chambers further comprises a wash chamber, a waste chamber, or a processing chamber adapted for dilution or reduction of salt concentration, or a combination thereof.

6. The system of claim 1, wherein the chambers of the at least one set of fluidic chambers are non-microfluidic chambers.

7. The system of claim 1, wherein fluid is selectively moved between the chambers of the at least one set of fluidic chambers using diaphragm valves.

8. The system of claim 1, wherein the disposable cartridge comprises a plurality of sets of fluidic chambers, and wherein the system is configured to perform the method on a plurality of samples in parallel.

9. The system of claim 1, which further comprises a processing module in fluidic communication with the reaction chamber and the separation and detection module and adapted to:

1) move the reaction product through a second microfluidic channel to a processing chamber;

2) process the reaction product; and

3) move the processed reaction product to the separation and detection module.

10. The system of claim 9, wherein processing the reaction product comprises diluting the reaction product or decreasing the salt concentration of the reaction product.

11. The system of claim 1, wherein the separation and detection module comprises an electrophoresis system comprising:

a) at least one electrophoresis sample channel having a channel inlet and a channel outlet;

b) at least one electrophoresis capillary having a capillary inlet and a capillary outlet, wherein the at least one electrophoresis capillary comprises an electrically conductive medium and is in communication with the at least one electrophoresis sample channel at a point of connection;

c) an anode and a cathode configured to apply a voltage across the capillary inlet and the capillary outlet of the at least one electrophoresis capillary, wherein one of the anode or the cathode comprises a first electrode comprising at least two forks, and wherein the at least two forks of the first electrode are in electrical communication with the at least one electrophoresis sample channel on different sides of the point of connection; and

d) a second electrode in electrical communication with the at least one electrophoresis sample channel and substantially opposite the capillary inlet of the at least one electrophoresis capillary at the point of connection.

12. The system of claim 1, wherein the separation and detection module comprises an optical system comprising:

a) an excitation source configured to direct excitation light to at least one fluorescent molecule, wherein the at least one fluorescent molecule emits light other than the excitation light when excited by the excitation light;

b) a rejection filter configured to filter out the excitation light and to allow transmission of the emitted light;

c) an imaging lens configured to focus the emitted light;

d) a dichroic mirror substantially transparent to the excitation light and configured to reflect the emitted light to a detector;

e) a focusing system comprising at least one lens configured to focus the light reflected from the dichroic minor; and

f) a photodetector configured to receive the focused light.

13. The system of claim 1, wherein the disposable cartridge comprises:

a) a microfluidic component comprising a plurality of intersecting microfluidic channels and at least one controllable valve configured to regulate flow of fluid between the intersecting microfluidic channels; and

b) a non-microfluidic component comprising a plurality of non-microfluidic chambers, wherein each non-microfluidic chamber is fluidically connected to at least one microfluidic channel;

wherein at least one valve regulates flow of fluid from at least one non-microfluidic chamber to another non-microfluidic chamber through a microfluidic channel.

14. The system of claim 1, which further comprises a cartridge cover and a pneumatic manifold, wherein:

the disposable cartridge can be releasably engaged with the cartridge cover and the pneumatic manifold;

the disposable cartridge comprises one or more pneumatically actuated valves and one or more microfluidic channels;

the pneumatic manifold and the cartridge cover are each fluidically connected to at least one pressure source; and

the pneumatic manifold and the cartridge cover are each adapted to control fluid flow in the disposable cartridge.

15. The system of claim 14, wherein the pneumatic manifold is adapted to actuate the one or more pneumatically actuated valves of the disposable cartridge and the cartridge cover is adapted to apply pressure to one or more chambers in the disposable cartridge.

16. The system of claim 1, which further comprises:

e) an actuator assembly configured to move fluid between chambers when the disposable cartridge is engaged with the actuator assembly;

f) a thermal cycler configured to cycle temperature in the reaction chamber adapted for thermal cycling when the disposable cartridge is engaged with the actuator assembly;

g) a capillary electrophoresis assembly configured to receive a reaction product from the disposable cartridge when the disposable cartridge is engaged with the actuator assembly and to perform capillary electrophoresis on the reaction product; and

h) a computerized control system configured to control the actuator assembly, the thermal cycler and the capillary electrophoresis assembly.

17. The system of claim 16, wherein the thermal cycler comprises a Peltier heating-and-cooling device.

18. The system of claim 1, which further comprises an article in computer-readable form and comprising code for operating the system.

19. The system of claim 1, wherein the plurality of STR markers comprises at least 5 STR markers.

20. The system of claim 1, wherein the plurality of STR markers comprises a plurality of CODIS STR markers.

21. The system of claim 20, wherein the plurality of STR markers comprises at least 5 CODIS STR markers.

22. The system of claim 20, wherein the plurality of CODIS STR markers is selected from the group consisting of D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPDX and FGA.

23. The system of claim 1, wherein the sample comprises a cell, a bodily fluid, blood, semen, urine, a buccal sample, saliva, a nasal sample, mucus, lung lavage, an amniotic sample, a vaginal sample, a fecal sample, a skin cell, or hair.

24. The system of claim 1, which is configured to perform the method in less than 2 hours.

25. The system of claim 1, which fits within an enclosure of no more than 10 ft³.