Devices and methods for the performance of miniaturized in vitro amplification assays

• US 6,706,519 B1
• Filed: 06/22/2000
• Issued: 03/16/2004
• Est. Priority Date: 06/22/1999
• Status: Expired due to Fees

• Current Assignee: Tecan Trading AG (Tecan Group AG)
• Original Assignee: Tecan Trading AG (Tecan Group AG)
• Inventors: Carvalho, Bruce L., Kellogg, Gregory J., Sheppard, Norman F., Arnold, Todd, Able, Charles, Lin, Hsin-Chiang, Kob, Mikayla, Kieffer-Higgins, Stephen, Ommert, Shari
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/602,394
• Time in Patent Office: 1,363 Days
• Field of Search: 435/286.5, 435/287.2, 435/287.3, 435/288.5, 435/288.7, 422/64, 422/67, 422/82.08
• US Class Current: 435/287.2
• CPC Class Codes: B01F 2215/0431 Numerical size values, e.g. diameter of a hole or conduit, area, volume, length, width, or ratios thereof
• CPC Class Codes: B01F 2215/0477 Numerical time values
• CPC Class Codes: B01F 33/3017 Mixing chamber
• CPC Class Codes: B01F 33/304 the mixing being performed in a mixing chamber where the products are brought into contact
• CPC Class Codes: B01F 35/712 for feeding fluids
• CPC Class Codes: B01F 35/7172 using capillary forces
• CPC Class Codes: B01F 35/71725 using centrifugal forces
• CPC Class Codes: B01F 35/71805 using valves, gates, orifices or openings
• CPC Class Codes: B01F 35/7547 using valves, gates, orifices or openings
• CPC Class Codes: B01F 35/896 characterised by the build-up of the device
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• 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: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 21/4795 spatially resolved investigating of object in scattering medium in vivo A61B
• 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

Abstract

This invention relates to methods and apparatus for performing microanalytic and microsynthetic analyses and procedures. The invention provides a microsystem platform and a micromanipulation device for manipulating the platform that utilizes the centripetal force resulting from rotation of the platform to motivate fluid movement through microchannels. The invention specifically provides devices and methods for performing miniaturized in vitro amplification assays such as the polymerase chain reaction. Methods specific for the apparatus of the invention for performing PCR are provided.

Claims

1. A centripetally-motivated microsystems platform for performing a DNA sample preparation comprising:

a) a rotatable platform comprising a substrate having a surface comprising one or a multiplicity of microfluidics structures embedded in the surface of the platform, wherein each microfluidics structure comprises

i) a sample input port fluidly connected to

ii) a cell lysis chamber in thermal contact with a temperature control element, wherein the cell lysis chamber comprises a filter element having a porosity that retains cell debris and precipitated proteins and nucleic acids in the chamber,

iii) a lysis buffer reservoir containing a lysis buffer, fluidly connected to the cell lysis chamber by a microchannel;

iv) a precipitation buffer reservoir, fluidly connected to the cell lysis chamber by a microchannel that comprises a sacrificial valve;

v) a DNA binding chamber, fluidly connected to the cell lysis chamber by a microchannel, wherein the microchannel is interrupted by a sacrificial valve, wherein the DNA binding chamber further comprises

vi) a DNA binding filter having a binding affinity for DNA;

vii) one or a multiplicity of wash buffer reservoirs, wherein each reservoir contains a wash buffer, and wherein each reservoir is fluidly connected to the DNA binding chamber by a microchannel that is interrupted by a sacrificial valve;

viii) a waste reservoir fluidly connected to the DNA binding chamber by a microchannel;

ix) a sample recovery chamber, fluidly connected to the DNA binding chamber by a microchannel interrupted by a sacrificial valve; and

x) an elution buffer reservoir, fluidly connected to the DNA binding chamber by a microchannel interrupted by a sacrificial valve;

wherein fluid flow through the microfluidic structures is motivated by centripetal force resulting from rotation of the platform and fluid flow through microchannels interrupted by sacrificial valves is dependent on the integrity of these valves.

2. A microsystem platform of claim 1 wherein the cell lysis chamber has a volumetric capacity of from about 50 μ

L to about 1000 μ

L.

3. A microsystem platform of claim 1 wherein the lysis buffer reservoir has a volumetric capacity of from about 25 μ

L to about 300 μ

L.

4. A microsystem platform of claim 1 wherein the precipitation buffer reservoir has a volumetric capacity of from about 35 μ

L to about 400 μ

L.

5. A microsystem platform of claim 1 herein each of the wash buffer reservoirs has a volumetric capacity of from about 50 μ

L to about 850 μ

L.

6. A microsystem platform of claim 1 wherein the elution buffer reservoir has a volumetric capacity of from about 20 μ

L to about 250 μ

L.

7. A microsystem platform of claim 1 wherein the waste reservoir has a volumetric capacity of from about 350 μ

L to about 1500 μ

L.

8. A microsystem platform of claim 1 wherein the DNA binding chamber has a volumetric capacity of from about 5 μ

L to about 20 μ

L.

9. A microsystem platform of claim 1 wherein the sample collection chamber has a volumetric capacity of from about 20 μ

L to about 250 μ

L.

11. A microsystems platform of claim 1 wherein the cell lysis chamber further comprises one or a multiplicity of mixing baffles.

12. A microsystems platform of claim 1 wherein the filter contained in the cell lysis chamber is positioned proximal to the microchannel connecting the cell lysis chamber to the DNA binding chamber, wherein fluid must flow through the filter to enter the microchannel.

13. A microsystems platform of claim 1 wherein the DNA binding filter contained in the DNA binding chamber is positioned proximal to the microchannel connecting the DNA binding chamber to the waste reservoir and the sample collection chamber, wherein fluid must flow through the filter to enter the microchannel.

14. A microsystems platform of claim 1 wherein the microchannel connecting the DNA binding chamber to the waste reservoir is the same microchannel that connects the DNA binding chamber to the sample collection chamber, wherein fluid flows through the portion of the microchannel fluidly connected to the waste reservoir only when the sacrificial valve interrupting the portion of the microchannel fluidly connecting the DNA binding chamber to the sample collection chamber is intact.

15. A microsystems platform according to claim 1, wherein each of the sacrificial valves is a wax valve that is in thermal contact with a heating element capable of producing sufficient heat to melt the wax and open the valve.

16. A microsystems platform according to claim 15, wherein each sacrificial valve further comprises a recrystallization chamber having a cross-sectional dimension sufficient to contain the wax comprising the wax valve and permit fluid flow through the microchannel.

17. A microsystems platform according to claim 1, wherein the cell lysis chamber is in thermal contact with a heating element.

18. A microsystems platform according to claim 17, further comprising an electric platen comprising a substrate bearing one or a multiplicity of temperature control elements, wherein each of the temperature control elements is electrically connected to at least two electrical leads, and wherein the electrical leads are connected to a power source through a slip ring;

wherein the substrate comprising the cell lysis chamber is separate from the platen and wherein the temperature control element is in thermal contact with the cell lysis chamber.

29. A microsystems platform according to claim 1, wherein the interior surfaces of the microfluidic structures are coated with parylene.

10. A microsystems platform of claim wherein the sample recovery chamber further comprises a sample outlet port.

19. A centripetally-motivated Microsystems platform for performing an in vitro amplification reaction comprising:

a) a rotatable platform comprising a substrate having a surface comprising one or a multiplicity of microfluidics structures embedded in the surface of the platform, wherein each microfluidics structure comprises

i) a sample chamber comprising a sample input port;

ii) a cell lysis buffer reservoir containing a cell lysis buffer;

iii) a neutralization buffer reservoir containing a neutralization buffer that is fluidly connected to a first reservoir by a microchannel;

iv) a first mixing microchannel fluidly connected to the sample buffer chamber and the cell lysis buffer reservoir, wherein the first mixing microchannel defines a longitudinal path in the surface of the platform having a length sufficient to mix the sample and the cell lysis buffer to a lysed cell mixture, wherein the first mixing microchannel is fluidly connected to

v) a second reservoir, wherein the first and second reservoirs are fluidly connected to

vi) a second mixing microchannel wherein the second mixing microchannel defines a longitudinal path in the surface of the platform having a length sufficient to mix the lysed cell mixture and the neutralization buffer to a DNA sample mixture, wherein the first mixing microchannel is fluidly connected to

vii) a third reservoir;

wherein the platform further comprises

viii) a fourth reservoir containing a solution comprising a DNA amplification reagent mixture, wherein the third and fourth reservoirs are fluidly connected to

ix) a third mixing microchannel wherein the third mixing microchannel defines a longitudinal path in the surface of the platform having a length sufficient to mix the DNA sample mixture and the DNA amplification reagent mixture to produce a DNA amplification reaction mixture, wherein the third mixing microchannel is fluidly connected to

x) a thermal cycling chamber in thermal contact with

xi) a temperature control element

fluid within the microchannels of the platform is moved through said microchannels 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 DNA amplification is performed in the thermal cycling chamber by alternating the temperature to denature template DNA, anneal primers and extend the primers with a polymerase.

20. A microsystem platform of claim 19 wherein the sample chamber has a volumetric capacity of from about 2 nL to about 1000 μ

L.

21. A microsystem platform of claim 19 wherein the cell lysis buffer reservoir has a volumetric capacity of from about 2 nL to about 1000 μ

L.

22. A microsystem platform of claim 19 wherein the neutralization buffer reservoir has a volumetric capacity of from about 2 nL to about 1000 μ

L.

23. A microsystem platform of claim 19 wherein each of the first, second, third and fourth reservoirs has a volumetric capacity of from about 2 nL to about 1000 μ

L.

24. A microsystem platform of claim 19 wherein the cell lysis buffer reservoir, the neutralization buffer reservoir and the third and fourth reservoirs each further comprise an input port.

25. A microsystem platform of claim 19 wherein each mixing microchannel comprises a plurality of bends having angles greater than 90°

.

26. A microsystem platform of claim 19 wherein the flow rate of fluid through each of the mixing microchannels is from about 1 nL/s to about 100 μ

L/s.

27. A microsystems platform of claim 19 wherein the thermal cycling chamber further comprises a sample outlet port.

28. A microsystems platform according to claim 19, further comprising an electric platen comprising a substrate bearing one or a multiplicity of temperature control elements, wherein each of the temperature control elements is electrically connected to at least two electrical leads, and wherein the electrical leads are connected to a power source through a slip ring;

wherein the substrate comprising the thermal cycling chamber is separate from the platen and wherein the temperature control element is in thermal contact with the thermal cycling chamber.

30. A microsystems platform according to claim 19, wherein the interior surfaces of the microfluidic structures are coated with parylene.