SAMPLE PROCESSING METHOD AND SAMPLE PROCESSING CARTRIDGE

• US 20140287955A1
• Filed: 10/11/2012
• Published: 09/25/2014
• Est. Priority Date: 10/11/2011
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Wende, Andy, Dahlke, Rainer, Himmelreich, Ralf, Rothmann, Thomas, Grosshauser, Gerd, Eberhard, Michael, Jeziorski, Markus, Attig, Hans, Drexler, Josef, Holzer, Eva
• Granted Patent: US 9,994,891 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 2200/0689 Sealing
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 3/5082 Test tubes per se
• 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: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present invention pertains inter alia to a method for analysing a sample comprising biomolecules, which comprises the following steps:

• A
  • a) lysing the sample to provide a lysed sample and optionally clearing the lysed sample;
  • b) contacting at least a portion of the lysed sample with a dry composition comprising reagents for performing the analytical method, thereby providing a reconstituted composition;
  • c) performing an analytical method using the reconstituted composition;
  • or
• B
  • a) contacting the sample with a lysis solution thereby providing a lysis mixture;
  • b) using the lysis mixture to reconstitute a dry composition comprising reagents for performing the analytical method, thereby providing a reconstituted composition;
  • c) performing an analytical method using the reconstituted composition,
  • wherein the reconstituted composition is optionally cleared and wherein subsequent to step b) at least one step is performed which supports the lysis of the sample.

Furthermore, specifically adapted processing cartridges and systems are provided.

Claims

1. A method for analysing a sample comprising one or more biomolecules, comprising:

A

a) lysing the sample to provide a lysed sample and optionally clearing the lysed sample;

b) contacting at least a portion of the lysed sample with a dry composition comprising one or more reagents for performing the analytical method, thereby providing a reconstituted composition;

c) performing an analytical method using the reconstituted composition;

or

B

a) contacting the sample with a lysis solution thereby providing a lysis mixture;

b) using the lysis mixture to reconstitute a dry composition comprising one or more reagents for performing the analytical method, thereby providing a reconstituted composition;

c) performing an analytical method using the reconstituted composition, wherein the reconstituted composition is optionally cleared and wherein subsequent to b), at least one step is performed which supports lysis of the sample.

2. The method according to claim 1, wherein clearing of the lysed sample and/or the reconstituted composition involves at least one of the following:

a) contacting the sample prior, during or after lysis with means for binding contaminants, optionally precipitates, that originate from the lysed sample;

b) passing at least a portion of the lysed sample or the reconstituted composition through a filter or membrane;

c) wherein said means for binding contaminants, optionally precipitates, according to 2 a) bind contaminants, optionally precipitates, that originate from the lysed sample;

d) binding of the contaminants occurs under conditions wherein contaminants originating from the lysed sample, optionally precipitates, bind to said means for binding contaminants according to 2a but wherein no substantial binding of the biomolecules of interest that are analysed in the analysis method occurs;

e) wherein the sample is contacted prior, during or after lysis with at least one solid support which binds to contaminates originating from the lysed sample, optionally precipitates, thereby forming a complex with the precipitates, and wherein said complex is optionally separated;

f) wherein the sample is contacted prior, during or after lysis with means for binding contaminants, optionally precipitates, wherein reconstitution of the dry composition occurs using the lysis mixture and wherein sample lysis is promoted by heating the reconstituted composition, whereby contaminants, optionally precipitates, that originate from the lysed sample and which are comprised in the reconstituted composition are bound, thereby providing a cleared reconstituted composition; and

/or

g) wherein clearing of the lysed sample and/or clearing of the reconstituted composition involves addition of at least one compound or composition that binds to and/or neutralizes one or more inhibitors of the analysis method.

3. The method according to claim 2, wherein the sample is contacted prior, during or after lysis with at least one solid support which binds to contaminates originating from the lysed sample, wherein the solid support has at least one of the following characteristics:

a) the solid support is selected from the group consisting of particles, plates and other particulate matter;

b) the solid support comprises a surface which binds contaminants, optionally precipitates, originating from the lysed sample to the solid support;

c) the solid support comprises metal oxides;

d) the solid support comprises a silica surface;

e) the solid support comprises one or more ligands on surface thereof to promote binding of contaminants to the solid support, wherein optionally, the ligands are selected from the group consisting of ion exchange groups, carboxyl groups, sulfonate groups and silane ligands;

f) the solid support comprises carboxyl groups on a surface thereof;

g) the solid support comprises or is a molecular sieve, optionally a zeolite;

h) incorporation of the solid support into the lysed sample and/or the reconstituted composition results in an elevation of pH value of the lysed sample and/or the reconstituted composition;

i) the solid support is magnetic; and

/or

j) the solid support is comprised in a lysis solution.

4. The method according to claim 1, wherein the reconstitution process has at least one of the following characteristics:

a) at least an aliquot of cleared lysed sample is contacted with the dry composition for reconstituting the dry composition;

b) at least an aliquot of lysis mixture is contacted with the dry composition for reconstituting the dry composition;

c) a predetermined amount of lysed sample, cleared lysed sample or lysis mixture is added to the dry composition for reconstitution;

d) a mixture comprising the dry composition and lysed sample, cleared lysed sample or lysis mixture is agitated to assist the reconstitution process; and

/or

e) a mixture comprising the dry composition and (i) lysed sample, (ii) cleared lysed sample or (iii) lysis mixture is stirred by aid of a magnet and a magnetic material is comprised in the dry composition to assist the reconstitution process.

5. The method according to claim 1, wherein lysis of the sample has at least one of the following characteristics:

a) lysis is achieved or assisted by one or more of heating, a treatment with one or more enzymes, the addition of one or more chemicals and/or a mechanical treatment of the sample;

b) lysis is achieved by contacting the sample with a lysis solution and optionally, lysis is assisted by heating;

c) for lysis of the sample at least one polymer is added which prevents or reduces an inhibition of the subsequent analytical method, optionally by unspecific complexing of potential inhibitors;

d) the lysis solution comprises a magnetic stirring bar;

e) the lysis solution comprises a solid support for clearing the lysate;

f) the lysed sample is cleared to provide a cleared lysed sample and wherein reconstitution of the sample involves contacting the dry composition with at least a portion of the cleared sample; and

/or

g) lysis is achieved and/or assisted by heating that is performed subsequent to reconstitution of the dry composition.

6. The method according to claim 1, wherein lysis of the sample comprises addition of a lysis solution which comprises

(a) at least one nonionic surfactant or mixture of non-ionic detergents,

(b) at least one polymer which prevents or reduces inhibition of a subsequent analytical method optionally by unspecific complexing of potential inhibitors;

(c) optionally a proteolytic enzyme,

(d) optionally a chelating agent for divalent cations and

(e) optionally a buffer substance.

7. The method according to claim 1, wherein lysis and reconstitution of the sample comprises:

a)

(i) contacting the sample with a lysis solution and a solid support for removing contaminants, optionally precipitates, thereby forming a lysis mixture;

(ii) heating the mixture to a temperature of at least 90°

C., optionally at least 95°

C.;

(iii) optionally cooling the lysed sample to a temperature below 50°

C.;

(iv) clearing the lysed sample by separating the formed complex comprising the solid support and precipitates thereby providing a cleared lysate; and

(v) using the cleared lysate for reconstituting the dry composition;

or

b)

(i) contacting the sample with a lysis solution and a solid support for removing contaminants, optionally precipitates, thereby forming a lysis mixture;

(ii) using the lysis mixture for reconstituting the dry composition;

(iii) heating the reconstituted composition to a temperature of at least 90°

C., optionally at least 95°

C.;

(iv) optionally cooling the reconstituted composition to a temperature below 50°

C.; and

(v) clearing the reconstituted composition by separating the formed complex comprising the solid support and precipitates thereby providing a cleared reconstituted composition;

or

c)

(i) contacting the sample with a lysis solution and a solid support for removing contaminants, optionally precipitates, thereby forming a lysis mixture;

(ii) using the lysis mixture for reconstituting the dry composition;

(iii) subjecting the reconstituted composition to an amplification reaction which comprises at least one heating step involving a temperature of at least 90°

C., optionally at least 95°

C.

8. The method according to of claim 1, wherein

the analytical method has at least one of the following characteristics:

a) said method involves analysis of biomolecules, optionally nucleic acids, comprised in the sample;

b) the analytical method comprises a detection reaction;

c) the analytical method comprises at least one heating step involving a temperature of at least 85°

C., at least 90°

C. or at least 95°

C.;

d) the analytical method comprises an amplification reaction;

e) the analytical method comprises a PCR or an isothermal amplification reaction;

f) the analytical method involves at least one heating step and lysis of the sample comprised in the reconstituted composition is assisted by said heating step; and

/or

g) the analytical method is performed in the presence of a solid support that is added for clearing;

and/or

the dry composition has at least one of the following characteristics

a) said composition is a freeze-dried composition;

b) said composition comprises at least some, optionally all, reagents necessary for conducting the intended analysis method;

c) said composition comprises at least some, optionally all, reagents necessary for conducting an amplification reaction;

d) said composition comprises one or more, optionally all, of the following reagents selected from the group consisting of a polymerase, a reaction buffer suitable for performing an amplification reaction, dNTPs, primers and/or probes; and

/or

e) said composition comprises a magnetic material that allows to assist a reconstitution process by enabling mixing of the composition during reconstitution by aid of a magnet.

9. The method according to claim 1, wherein the biomolecules are nucleic acids, the dry composition is a freeze-dried composition and the lysed sample or the reconstituted composition is cleared.

10. A processing cartridge suitable for use in a method for analysing a sample comprising biomolecules according to claim 1, wherein the processing cartridge comprises a cartridge body and at least one reaction chamber which comprises a dry composition comprising reagents for performing an analytical method, wherein the cartridge body comprises a sample intake opening, at least one sample outlet and a fluid passageway that connects the sample intake opening and the sample outlet, wherein the sample outlet opens into the reaction chamber, wherein the cartridge is designed such that reconstitution of the dry composition comprised in the reaction chamber is achieved by a sample that enters the cartridge through the sample intake opening.

11. The processing cartridge according to claim 10, having at least one of the following characteristics:

a) the fluid passageway comprises at least one of channels and/or cavities and is at least partially formed into the surface of the cartridge body;

b) the cartridge body comprises a system of channels and/or cavities for a predefined sample passageway after the sample has entered through the sample intake opening;

c) the cartridge comprises at least two reaction chambers, and wherein the fluid passageway for the sample branches and ends in at least two sample outlets, wherein each sample outlet (5) opens into a different reaction chamber;

d) the one or more reaction chambers are integrally comprised in the cartridge body;

e) the one or more reaction chambers are provided as separate device(s) that is/are assembled to the cartridge body;

f) the cartridge body comprises means, preferably a connection projection, for connecting at least one reaction chamber to the cartridge body;

g) the cartridge body comprises a plurality of reaction chambers and/or a plurality of means, preferably connection projections, for connecting the reaction chambers;

h) the cartridge body comprises means, preferably a connection projection, for connecting a sample vessel to the cartridge body;

i) the cartridge body comprises a fluid intake opening B which is through a fluid passageway B in fluid communication with the sample vessel if said vessel is assembled to the cartridge body and wherein air is introduced into the sample vessel via the fluid outlet B; and

/or

j) the cartridge body comprises a tube 30) which is located within the sample vessel if said vessel is assembled to the cartridge body and wherein the sample reaches the sample intake opening via said tube.

12. The processing cartridge according to claim 10, wherein means are provided for controlling the amount of liquid that can enter the reaction chamber for reconstitution of the dry composition.

13. The processing cartridge according to claim 10, wherein the reaction chamber and/or the means for connecting the reaction chamber to the cartridge body comprise at least one fluid opening A through which a fluid optionally air can exit the reaction chamber.

14. The processing cartridge according to claim 13, wherein the fluid opening A through which a fluid can exit the reaction chamber comprises a barrier, wherein said barrier substantially prevents the passage of liquid and wherein, optionally, said barrier has at least one of the following characteristics:

a) said barrier allows passage of air at least before said barrier comes into contact with a liquid but wherein said barrier substantially prevents passage of liquid;

b) said barrier is a membrane or filter;

c) said barrier is hydrophobic; and

/or

d) said barrier is porous.

15. The processing cartridge according to claim 10, wherein

the sample outlet and/or the fluid opening A are designed as tube which is at least partially comprised in a side wall of a reaction chamber and/or in a side wall of the means for connecting the reaction chamber to the cartridge

and/or

the cartridge body comprises a fluid outlet A that is connected to the fluid opening A by a fluid passageway A.

16. The processing cartridge according to claim 10, comprising means that allow to close the sample passageway and/or the fluid passageway A.

17. The processing cartridge according to claim 16, wherein at least one barrier is provided within the sample passageway and/or the fluid passageway A, which in conjunction with a lid that covers said fluid passageway allows to close the sample passageway and/or the fluid passageway A if pressure is applied to the lid.

18. The processing cartridge according to claim 10, wherein the cartridge body is assembled to a sample vessel comprising a sample vessel opening that is arranged in such a manner that the sample vessel opening is in fluid connection with the sample intake opening.

19. The processing cartridge according to claim 18, wherein the sample vessel contains a sample to be analysed mixed with a lysis composition and wherein, optionally, the lysis composition has at least one of the following characteristics:

a) said lysis composition is a lysis solution which comprises

(a) at least one nonionic surfactant or mixture of non-ionic detergents,

(b) at least one polymer which prevents or reduces inhibition ofa subsequent analytical method optionally unspecific complexing of potential inhibitors;

(c) optionally a proteolytic enzyme,

(d) optionally a chelating agent for divalent cations and

(e) optionally a buffer substance;

b) said lysis composition comprises a magnetic stirring bar; and

/or

c) said lysis composition comprises a solid support for clearing lysate.

25. The processing cartridge according to claim 10, comprising a cartridge body.

26. An operating method capable of being used for performing an analysis method using a system for performing an analytical method of a sample comprising biomolecules, wherein said system comprises

a) a processing cartridge according to claim 10;

wherein the processing cartridge comprises at least one reaction chamber comprising a dry composition comprising reagents for the analytical method;

b) a sample vessel for receiving a sample comprising biomolecules, wherein the vessel can be assembled to the processing cartridge;

c) a processing device for receiving the processing cartridge comprising the vessel assembled thereto and for performing the analysis method in conjunction with the processing cartridge;

and wherein the method comprises

a) connecting a sample vessel comprising a sample to the processing cartridge;

b) inserting the processing cartridge into a processing device; and

c) starting a fully automatic assay.

20. Cartridge body suitable for providing a fluid connection from a sample vessel that contains a fluid and can be assembled to the cartridge body, to at least one reaction chamber that can be assembled to the cartridge body or which is provided as integral part of the cartridge body, comprising

a sample intake opening and a sample outlet,

a sample passageway that connects the sample intake opening and the sample outlet,

a sample vessel connection projection for connecting the sample vessel, wherein upon assembly of the sample vessel to said connection projection the sample intake opening is in fluid communication with the sample vessel,

at least one reaction chamber connection projection for connecting a reaction chamber wherein upon assembly of the reaction chamber to said connection projection the sample outlet is in fluid communication with the reaction chamber and/or comprising at least one reaction chamber that is provided as integral part of the cartridge body

wherein at least one fluid opening A is provided in the reaction chamber connection projection and/or in the reaction chamber and wherein said fluid opening comprises a barrier, wherein said barrier allows the passage of air at least before said barrier comes into contact with a liquid but wherein said barrier substantially prevents the passage of liquid.

21. The cartridge body according to claim 20, wherein said barrier has at least one of the following characteristics:

a) said barrier is a membrane or filter;

b) said barrier is hydrophobic; and

/or

c) said barrier is porous.

22. The cartridge body according to claim 20, having at least one of the following characteristics:

a) the sample outlet and/or the fluid opening A are designed as tube wherein said tube is at least partially comprised in a side wall of a reaction chamber and/or in a side wall of the reaction chamber connection projection,

b) the cartridge body comprises a fluid outlet A and a fluid passageway A that connects the fluid opening A and the fluid outlet A; and

/or

c) the cartridge body comprises means that allow to close the sample passageway and/or the fluid passageway A.

23. The cartridge according to claim 20, comprising

a fluid intake opening B and a fluid outlet B,

a fluid passageway B that connects the fluid intake opening B and the fluid outlet B,

wherein upon assembly of the sample vessel to the sample vessel connection projection the fluid outlet B and the sample intake opening are in fluid communication with the sample vessel.

24. Cartridge body according to claim 20, having at least one of the following characteristics:

a) at least one barrier is provided within the sample passageway and/or the fluid passageway A, which in conjunction with a lid that covers said fluid passageway allows to close the sample passageway and/or the fluid passageway A if pressure is applied to the lid;

b) the sample outlet and/or the fluid opening A, optionally both, are designed as tube wherein said tube is at least partially comprised in a convexity of a side wall of a reaction chamber and/or in a convexity of a side wall of the reaction chamber connection projection;

c) the sample vessel connection projection comprising side walls and a base wall and optionally comprising an opening opposite the base wall, wherein the fluid outlet B and the sample intake opening are arranged in the base wall or at least one of the side walls;

d) the reaction chamber connection projection comprising side walls and a base wall, wherein the sample outlet and optionally the fluid opening A are arranged in the base wall or at least one of the side walls;

e) the cartridge body comprises a tube with a first end opening and a second end opening, whereby the first end opening is aligned with the sample intake opening, wherein optionally the tube is a rigid, longitudinal tube;

f) the side walls of the sample vessel connection projection and/or the side walls of the reaction chamber connection projection form a tubular body;

g) the side walls of the sample vessel connection projection are adapted to fit into an opening of the sample vessel, wherein, preferably, the side walls of the sample vessel connection projection form a tubular body and that the tubular body has an external thread and/or the side walls of the reaction chamber connection projection are adapted to fit into an opening of a reaction chamber that can be assembled to said projection, wherein, optionally, the side walls of the reaction chamber connection projection form a tubular body and that the tubular body has an external thread;

h) the sample passageway is at least partially provided by a channel or cavity formed into the surface of the cartridge body and/or the fluid passageway A is at least partially formed by a channel or cavity formed into the surface of the cartridge body and/or the fluid passageway B is at least partially provided by a channel or cavity formed into the surface of the cartridge body;

i) a lid is attached to the cartridge body, wherein the lid closes the channel or cavity that forms at least part of the sample passageway, the fluid passageway A and/or the fluid passageway B, wherein said lid optionally is a foil;

j) the sample passageway is at least partially formed by a channel or cavity formed into the surface of the cartridge body and the fluid passageway A is at least partially formed by a channel or cavity formed into a surface of the cartridge body and wherein the fluid passageway B is at least partially formed by a channel or cavity formed into the surface of the cartridge body, whereby a lid is attached to the cartridge body that closes the said channels or cavities;

k) the fluid intake opening B, the fluid outlet B, the sample intake opening, the sample outlet, the fluid opening A and the optional fluid outlet A are arranged in the same surface of the cartridge body;

l) the fluid opening A, the fluid intake opening B and/or the optional fluid outlet A is closed by a membrane or a filter;

m) the cartridge body comprises at least one additional reaction chamber connection projection for connecting a further reaction chamber to the cartridge body, wherein said further reaction chamber connection projection comprises the same elements as the reaction chamber connection projection, and wherein the sample outlet of said further reaction chamber connection projection is in fluid communication with the sample passageway; and

/or

n) the cartridge body comprises at least one additional reaction chamber that is in fluid communication with the sample passageway.