HYDROPHOBICITY BASED FLOW PREVENTION IN SAMPLE PREPARATION

Abstract

The present invention relates to a cartridge comprising or consisting of, from top to bottom, the following elements: (a) an inlet; (b) a top volume; (c) at least one optional layer made of a first material; (d) adjacent to (b) or, if present, to (c), at least one layer of chromatographic material; (e) adjacent to (d) at least one layer made of first material; and (f) an outlet; wherein said first material is hydrophobic and porous.

Claims

1. A cartridge comprising or consisting of, from top to bottom, the following elements: (a) an inlet; (b) a top volume; (c) at least one optional layer made of a first material; (d) adjacent to (b) or, if present, to (c), at least one layer of chromatographic material; (e) adjacent to (d) at least one layer made of first material; and (f) an outlet; wherein said first material is hydrophobic and porous.

2. The cartridge of claim 1, wherein the layers of (e) and, if present, of (c) prevent the flow of a polar liquid with a surface tension of at least 35 mN/m, at least 50 mN/m or at least 70 mN/m at ambient pressure and allow the flow of said polar liquid through said disk(s) at elevated pressure, and/or when vacuum and/or centrifugal force is applied.

3. The cartridge of claim 2, wherein said elevated pressure is at least 10 000 Pa above ambient pressure, preferably at least 100 000 Pa above ambient pressure, or a consequence of centrifugation with at least 50 g.sub.n or at least 500 g.sub.n, wherein said elevated pressure may be exerted from above and/or as vacuum from below, and wherein preferably said polar liquid is water.

4. The cartridge of any one of the preceding claims, wherein (a) the pores of said first material have a width of between about 1 nm and about 20 m, preferably between about 0.01 m and about 5 m, most preferred between about 0.22 m and about 1 m; (b) the contact angle of water on a surface of said first material is at least 90 degrees, preferably at least 100 degrees, more preferably at least 110 degrees; and/or (c) the surface energy of said first material is 70 mN/m or less, preferably 50 mN/m or less or 30 mN/m or less, more preferably about 20 mN/m or less.

5. The cartridge of any one of claims 1 to 4, wherein said first material of (e) and, if present, of (c), are independently selected (a) from polytetrafluorethylene (PTFE), perfluoroalkoxy alkane (PFA), and fluorinated ethylene propylene (FEP), wherein preferably said material is provided as a membrane (b) C18 material, C8 material, C4 material and benzene, wherein preferably said first material is bound to beads or to a membrane, and wherein more preferably said layer(s) is/are Empore SDB-XC extraction disks; and (c) superhydrophobic particles made of manganese oxide polystyrene (MnO.sub.2/PS) nano-composite, zinc oxide polystyrene (ZnO/PS) nano-composite, precipitated calcium carbonate, carbon nano-tube structure, and/or silica, wherein preferably said particles form a coating of said layer(s) of chromatographic material.

6. The cartridge of any one of the preceding claims, wherein the segment of said cartridge comprising or consisting of elements (c), (d) and (e) has constant width and/or is cylindrical, wherein preferably said segment further comprises or further consists of element (b), and wherein more preferably said segment further comprises or further consists of elements (a) and/or (f).

7. The cartridge of any one of the preceding claims, wherein said layer of (c) is present, thereby rendering said top volume (b) a reaction volume.

8. Use of the cartridge of any one of the preceding claims for controlling flow of polar liquid through the chromatographic material of (d).

9. Use of the cartridge of any one of the preceding claims for preventing backflow of polar liquid from below the disk of (e) such as backflow of water from a water bath.

10. Use of the cartridge of any one of the preceding claims for storage of polar liquids, polar liquids preferably being biological samples, biological samples preferably comprising peptides, polypeptides and/or proteins.

11. Use of the cartridge of any one of the preceding claims, wherein said disk of (c) is present, for retaining polar liquids above said disk of (c).

12. Use of the cartridge of any one of the preceding claims for preventing the chromatographic material of (d) from drying, wherein drying is the loss of a polar liquid.

13. A method of sample preparation, said method comprising: (a) transferring a sample to a cartridge of any one of the preceding claims via the inlet of said cartridge; and (b) applying pressure, vacuum and/or centrifugal force; thereby preparing said sample.

14. The method of claim 13, furthermore comprising one or both steps (aa) and (bb): (aa) after step (a) and prior to step (b), adding one or more reagents and/or allowing (a) reaction(s) to occur, wherein preferably said layer (c) of said cartridge is present; and (bb) collecting the eluate flowing from the outlet of said cartridge, optionally after changing conditions, said collecting optionally comprising fractionating.

15. A kit comprising or consisting of (a) a cartridge according to any one of the preceding claims; and (b) (i) a protease, preferably trypsin and/or Lys-C; an alkylating agent, preferably chloroacetamide; a reducing agent, preferably a phosphine-based reducing agent; a standard for mass-spectrometric analysis; a chaotropic agent, preferably GdmCl, a detergent, preferably SDC; and/or means for establishing a pH-value in said container of between 7 and 9, preferably 8 and 9, more preferably 8.5; (ii) a nuclease, preferably an endonuclease; and/or reagents for nucleic acid amplification, preferably by PCR; and/or (iii) one or more buffers for loading, washing, and eluting of analytes of the chromatography material.

Description

[0114] The figures show:

[0115] FIG. 1: Cartridge of the invention with hydrophobic layers and a constant cross-section design. (1) Inlet and reaction volume (implementing items (a) and (b) of the main embodiment). (2) Top hydrophobic layer to prevent flow into chromatographic material of choice (implementing item (c)). (3) Chromatographic material (implementing item (d)). (4) Bottom hydrophobic layer to prevent backflow into chromatographic material from the bottom outside (implementing item (e)). (6) Outlet equipped with a grid (implementing item (f)).

[0116] FIG. 2: Cartridge of the invention with hydrophobic layers and a design similar to a syringe. (1) Inlet and reaction volume. (2) Top hydrophobic layer to prevent flow into chromatographic material of choice. (3) Chromatographic material. (4) Bottom hydrophobic layer to prevent backflow into chromatographic material from the bottom outside. (5) Outlet and bottom volume.

[0117] FIG. 3: Cartridge of the invention with a simple grid at the bottom.

[0118] The examples illustrate the invention.

EXAMPLE 1

Water Retention

Materials and Methods

[0119] Various discs of alternating chemistries were tested for water retention (Tab. 1). For this purpose, discs of 15 mm.sup.2 were filled into cartridges of a fill volume of max. 300 l. The cartridges were filled with 300 l ultrapure LC-MS grade water and were centrifuged at room temperature (24 C.) with alternating speed and time settings (Tab. 2). The flow-through was quantified after centrifugation to determine the efficiency of retention.

TABLE-US-00001 TABLE 1 Materials used for testing. Descrip- Hydropho- Materials Manufacturer tion Discs bicity Wettability 1 3M SDB-XC 1 +++ 2 3M SDB-RPS 1 + +++ 3 Piper Filter PTFE 1 +++ GmbH 1-2 m 4 Piper Filter PTFE 1 +++ GmbH 5-6 m 5 Piper Filter PTFE 2 +++ GmbH 1-2 m 6 3M Cation 1 +++ 7 3M Anion 1 +++ 8 3M Activated 1 +++ Carbon 9 3M C8 1 ++ 10 3M C18 1 +++

TABLE-US-00002 TABLE 2 Centrifugation settings for testing. 300 g.sub.n corresponds to 0.6 bar pressure difference across the membrane. Condition Acceleration [g.sub.n] Time [s] Temp. [ C.] A 100 60 24 B 300 60 24 C 500 60 24 D 1000 60 24 E 1000 90 24 F 300 600 24 G 300 3600 24

Results and Discussion

[0120] All hydrophobic materials tested for short/high speed centrifugation displayed better liquid retention at low centrifugation speed. Highly hydrophobic and thick membranes (materials 1 and 5) display very good retention for short times at accelerations of up to 1,000 g.sub.n (corresponds to a pressure difference across the membrane of 1.9 bar; see Tab. 4 for the amount of liquid passing through the cartridge under the given conditions). More hydrophilic material (material 2) displays higher liquid retention at lower speed but a higher flow at higher speeds.

TABLE-US-00003 TABLE 4 Short centrifugation of few selected materials. Condition 1 2 3 4 5 A 0 l 0 l 0 l 0 l 0 l B 0 l 10 l 0 l 100 l 0 l C 0 l 150 l 150 l 150 l 0 l D 2 l 290 l 200 l 200 l 200 l E 10 l 300 l 280 l 280 l 280 l

[0121] To test the overall retention over longer time periods longer centrifugation steps were tested with a broader spectrum of materials (Tab. 5). All highly hydrophobic materials except for PTFE 5-6 m retain water at 300 g.sub.n for 1 h at room temperature. SAX (material 7) displayed delayed flow but did not withstand the long-term exposure to 300 g.

TABLE-US-00004 TABLE 5 Long term centrifugation. Condition 1 2 3 4 5 6 7 8 9 10 F 0 300 0 300 0 300 10 0 0 0 l l l l l l l l l l G 0 300 0 300 0 300 300 0 0 0 l l l l l l l l l l

[0122] Liquid retention was successfully achieved at centrifugation speeds up to 300 g.sub.n with hydrophobic materials and filtration pores below 5 m. Large pores such as 5 m would therefore need a higher surface energy as can be achieved with superhydrophobic materials or coatings. The backpressure of some materials (material 2 and 7) may delay the liquid flow across these hydrophilic membranes however all hydrophilic membranes did not retain water for extended periods of time. Hydrophobic membranes can therefore be used to selectively allow flow at predetermined flow forces and thereby control the entire procedure.

EXAMPLE 2

Chromatography

Materials and Methods

[0123] Standard constructions for SCX and SAX chromatography were tested with and without a PTFE 1-2 m membrane as bottom layer with and with and without PTFE 1-2 m as top layer (Tab. 3). For this purpose, discs of 15 mm.sup.2 were filled into cartridges of a fill volume of max. 300 l. The cartridges were filled with 300 l 1% acetic acid (AcOH) for SCX and 100 mM sodium hydroxide (NaOH) for SAX chromatography. To test liquid retention and selective loading, the cartridges were centrifuged at 500 g.sub.n for 1 min (PL1) and 10 min (PL2) at room temperature. Subsequently the cartridges were centrifuged at 3,000 g.sub.n for 1 min for analyte loading (L). The cartridges were then washed twice with 300 l 0.1% AcOH for SCX and 10 mM NaOH for SAX chromatography at 3,000 g.sub.n for 1 min (W1, W2). The sample was then eluted with 300 l 1% ammonium hydroxide (NH4) for SCX and 1% AcOH for SAX chromatography at 3.000 g.sub.n for 1 min (E).

TABLE-US-00005 TABLE 3 Materials used for testing. Layers numbered from top to bottom. Materials Layer 1 Layer 2 Layer 3 11 Cation (SCX) 12 Anion (SAX) 13 Cation (SCX) PTFE 1-2 m 14 Anion (SAX) PTFE 1-2 m 15 PTFE 1-2 m Cation (SCX) PTFE 1-2 m 16 PTFE 1-2 m Anion (SAX) PTFE 1-2 m

Results and Discussion

[0124] All combinations of membranes with a PTFE 1-2 m (materials 13-16) retained the liquid at centrifugation at 500 g.sub.n for 10 min while the cartridges with chromatography material only did not retain the liquid even after 1 min at 500 g.sub.n centrifugation (materials 11, 12).

TABLE-US-00006 TABLE 6 Selective SCX and SAX chromatography flow using hydrophobic membranes to retain liquid. Condition PL1 PL2 L W1 W2 E 11 300 l 300 l 300 l 300 l 300 l 300 l 12 300 l 300 l 300 l 300 l 300 l 300 l 13 0 l 0 l 300 l 300 l 300 l 300 l 14 0 l 0 l 300 l 300 l 300 l 300 l 15 0 l 0 l 300 l 300 l 300 l 300 l 16 0 l 0 l 300 l 300 l 300 l 300 l

[0125] SCX and SAX chromatography can be selectively performed using predetermined centrifugation speeds without risk of prior flow. The flow can be controlled by centrifugation and can therefore be used to define which sample flows across the chromatography material at which stage.