Abstract
A device and method for separation of components of a sample, in particular for pressure separation of immiscible or liquid systems with limited miscibility having at least one first chamber with a U- or V-shaped bottom wherein at least one aperture with a diameter within the range of 1 to 100 μm, preferably 1 to 40 μm, is provided in the first chamber and at least the surface of each aperture is hydrophilized or hydrophobized is disclosed. The device further has a second chamber surrounding the outside of the bottom of the first chamber. The invention also provides a method for separating components of a sample using this device and additionally enables parallel arrangement for plurality of separating conditions and serial arrangement for plurality of separated samples at the same time.
Claims
1: A device for separation of components of a sample, comprising at least one first chamber (11, 21b, 41, 51b, 61) with a U- or V-shaped bottom, wherein at least one aperture (13a, 13b, 23b, 43, 53b, 63) with a diameter within the range of 1 to 100 μm, preferably 1 to 40 μm, is provided in the first chamber and wherein at least the surface of each aperture (13a, 13b, 23b, 43, 53b, 63) is hydrophilized or hydrophobized, wherein the device further comprises a second chamber (12, 22, 42, 52, 62) surrounding the outside of the bottom of the first chamber.
2: The device according to claim 1, wherein the first chamber is closable, preferably by a lid, a membrane or a foil.
3: The device according to claim 1, wherein the surface of the aperture (13a, 13b, 23b, 43, 53b, 63) as well as the surface of the bottom of the first chamber (11, 21b, 41, 51b, 61) are hydrophilized or hydrophobized, wherein the bottom is at least the inner surface area of the first chamber in which the said at least one aperture is located; or the surface of the aperture (13a, 13b, 23b, 43, 53b, 63) as well as the inner surface of the first chamber (11, 21b, 41, 51b, 61) are hydrophilized or hydrophobized.
4: The device according to claim 1, wherein the material of the chambers is plastic, preferably selected from polycarbonate; polyolefins such as polyethylene, polypropylene; polystyrene; polyvinyl chloride; and fluorinated polymers; such as polytetrafluoroethylene.
5: The device according to claim 1, which is provided with at least one ventilation opening (14, 24, 34a, 34b, 34c, 34d, 64) located in the first and/or second chamber of the device; preferably the ventilation opening (14, 24, 34a, 34b, 34c, 34d, 64) is located between the edge furthest from the bottom of the chamber and half of the distance between the bottom and the edge of the chamber.
6: The device according to claim 1, wherein the device contains a plurality of first chambers (41, 51a, 51b) and a plurality of second chambers (42, 52), wherein preferably the first chambers (41, 51a, 51b) are arranged in a first holder to form a first chamber system and second chambers (42, 52) in a second holder to form a second chamber system, and the first chamber system is inserted into the second chamber system.
7: The device according to claim 1, wherein the device contains a plurality of first chambers (21a, 21b, 51a, 51b) inserted into each other, so that in N first chambers, each of (N−1) first chambers surrounds the outside of the bottom of the preceding first chamber in the direction of the flow of liquids through the device, and the outside of the bottom of the last first chamber is surrounded by a second chamber (22, 52).
8: The device according to claim 7, wherein at least one of the first chambers is the first chamber (21b, 51b) having at least an aperture (23b, 53b) with a hydrophilized or hydrophobized surface, and wherein the further first chambers (21a, 51a) are chambers having a V-shaped or U-shaped bottom containing at least one aperture with a diameter in the range of 1 to 100 μm, preferably 1 to 40 μm, wherein the said further first chambers (21a, 51a) may not have any surface adjustment, or may have at least part of the inner surface, preferably at least the surface of the aperture (23a, 53a), modified by separating means the embodiment of the device suitable for binding at least one component of a sample.
9: The device according to claim 8, wherein the separating means are selected from antibodies, affinity agents, hydrophobic agents, hydrophilic agents, ionic agents, chelating agents, magnetic components, components based on imprinted polymers, and combinations thereof.
10: The device according to claim 6, wherein the holder(s) with the first chambers and the holder with the second chambers are multi-well plates, provided with apertures in the bottoms of the wells representing the first chambers, and the plates are arranged so that the wells representing the second chambers surround the outside of the bottom of the wells representing the first chambers.
11: The method of sample separation in a liquid-liquid system using the device according to claim 1, containing the following steps: introducing a system containing immiscible liquids into a first chamber with a hydrophilized or hydrophobized surface of at least the aperture of the first chamber, or into an arrangement of a plurality of first chambers, wherein at least one of the first chambers has a hydrophilized or hydrophobized surface of at least the aperture, introducing a fluid sample (i.e. a liquid sample or a gas sample) into the first chamber; this step can be performed together with the step of introducing the system containing immiscible liquids or subsequently to the step of introducing the system containing immiscible liquids, or prior to the step of introducing the system containing immiscible liquids, optional step of emulsification (e.g. by sonication bath) and subsequent phase stabilization (especially when the fluid sample is introduced separately (prior to or subsequently) from the system containing immiscible liquids), application of pressure force on the system in the first chamber causing the liquid fraction having the same chemical hydrophilic or hydrophobic nature, respectively, as the hydrophilic or hydrophobic nature of the aperture surface, respectively, to pass through the aperture into the next chamber, and causing the retention of the liquid fraction having the opposite chemical hydrophilic or hydrophobic nature, respectively, than the hydrophilic of hydrophobic nature of the aperture surface, respectively.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0053] FIG. 1 schematically shows a basic embodiment of the device with one first chamber and one second chamber.
[0054] FIG. 2 schematically shows an example of a serial arrangement of the device with a plurality of first chambers and one second chamber.
[0055] FIG. 3 schematically shows examples of the location of the ventilation opening.
[0056] FIG. 4 schematically shows an example of a parallel arrangement of the device with the same number of first and second chambers.
[0057] FIG. 5 schematically shows an example of a parallel arrangement of serial arrangements of chambers.
[0058] FIG. 6 schematically shows separation of a mixture of three immiscible liquids in a device shown in FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
[0059] Examples of various embodiments of the device according to the invention are shown in FIGS. 1 to 6. FIG. 1 shows a basic embodiment of the device with a first U-shaped chamber 11, the bottom of the first chamber is surrounded by a second chamber 12. The first chamber 11 has an aperture 13a located at the lowest point of the U-shape if intended for use for centrifugation using a swinging rotor and/or an aperture 13b located at a point in the surface of the upper chamber where the pressure force is highest during centrifugal rotor centrifugation when intended for use in centrifugation using an angular rotor. The aperture size typically ranges from 1 to 100 μm in diameter. At least the surface of the aperture is hydrophilized or hydrophobized. Hydrophilization or hydrophobization means a surface modification or incorporation of material, fabric and/or compound in order to increase hydrophilicity, respectively hydrophobicity of the initial material. At least one ventilation opening 14 may be present in the second chamber 12 when the compensation of pressure changes caused by the flow of a sample fraction into this chamber during the separation is required. In this embodiment, the first chamber 11 is not sealed, therefore the pressure equalizes due to open top of the first chamber and a ventilation opening is not needed.
[0060] FIG. 2 shows an example of a serial arrangement of the device with first chambers 21a, 21b which are provided with apertures 23a and 23b. The upper first chamber 21a is inserted into the lower first chamber 21b, and the lower first chamber 21b is inserted into the second chamber 22. The second chamber 22 is provided with a ventilation opening 24. In the upper first chamber 21a is a solid sorbent 25, while the lower first chamber 21b has a hydrophilized or hydrophobized at least the surface of the aperture 23b. FIG. 3 shows examples of the ventilation opening location. In embodiment A, the ventilation opening 34a is located in the sidewall of the first chamber, which is in this embodiment closed by a lid, and another aperture 34b is provided in the second chamber. In embodiment B, the upper chamber is also closed with a lid, and a ventilation opening 34c is provided in the lid, and another ventilation opening 34d is provided in the sidewall of the second chamber.
[0061] FIG. 4 shows schematically an example of a parallel arrangement of the device with first chambers 41 with apertures 43 and second chambers 42. The chambers may, for example, be constructed as described in FIG. 1.
[0062] FIG. 5 shows schematically an example of a parallel arrangement of serial arrangements of chambers, wherein upper first chambers 51a with apertures 53a are arranged in parallel, with a corresponding number of lower first chambers 51b with apertures 53b also arranged in parallel, and with second chambers 52 arranged in parallel. In the upper first chambers 51 are provided particles of solid sorbent (representing any other separating method mentioned above), and the lower first chambers 51b have at least the surface of the aperture hydrophilized or hydrophobized.
[0063] FIG. 6 schematically shows the separation of a mixture of three immiscible liquids on a device according to FIG. 1. Immiscible liquids 66, 67, 6 are placed in a first chamber 61 having at least the surface of an aperture 63 hydrophilized or hydrophobized. Thus, only a liquid of the same chemical nature can pass through the aperture 63 (i.e. a hydrophilic liquid passes through a hydrophilized aperture, and a hydrophobic liquid passes through a hydrophobized aperture) under the action of a pressure force. The liquid passing through the aperture 63 of the first chamber flows into the second chamber 62, where it is captured and retained. The ventilation opening 64 equalizes pressure in the second chamber 62 with the pressure of the surrounding environment (if required). Without the ventilation opening 64, the pressure in the second chamber would increase due to the volume of liquid incoming from the first chamber 61.
EXAMPLES
Example 1
[0064] The bottom of the first chamber 11 of the device according to FIG. 1, wherein the first chamber is formed by a polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture with external dimensions of 20×2 μm. The thus prepared capillary aperture 13a was then hydrophobized by silanization (pressure perfusion of the capillary aperture with 100 μl solution of dimethyldichlorosilane in 1,1,1-trichloroethane). After drying (24 h, at RT), the bottom of the first chamber 11 was covered with Parafilm foil and 10 μl of a solution containing liposoluble Sudan B dye (Sigma, 0.1 mg/ml) in chloroform and 170 μl of PBS (saline, phosphate buffered saline, 140 mM NaCl, 10 mM HEPES, pH 7.4) was added. The first chamber closed with a lid (according to FIG. 3) was vortexed for 1 min. Subsequently, the Parafilm foil was removed from the first chamber 11 of the device, and the first chamber was then inserted into the second chamber 12 provided with a ventilation opening 34b for pressure equalization (according to FIG. 3). The embodiment of the device according to FIG. 1 was centrifuged at room temperature for 3 minutes at a centrifugal force of 100×g in a swinging rotor. The inspection revealed that 5 μl of Sudan B solution in chloroform had passed into the second chamber 12 and the colorless aqueous phase remained quantitatively in the first chamber 11. Spectrophotometric measurements at 600 nm on Nanodrop confirmed that the chloroform phase in the second chamber contained 98% of Sudan B.
Example 2
[0065] The bottom of the first chamber 11 of the device according to FIG. 1, wherein the first chamber is formed by a polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture with external dimensions of 20×2 μm. The thus formed capillary aperture 13a was then hydrophobized by silanization (pressure perfusion of the of the capillary aperture with 100 μl solution of dimethyldichlorosilane in 1,1,1-trichloroethane). After drying (24 h, at RT), embodiment of the device according to FIG. 1 was utilized for extraction of cobalt ions in complex with 1-(2-pyridylazo)-2-naphthol (PAN, Sigma) from an aqueous solution. 170 μl of a 0.25M aqueous solution of sodium nitrate containing 30 μg/l CoCl.sub.2 was added to 0.5 μl of a 0.001M aqueous solution of a cobalt chelator—PAN. After a few minutes, the solution turned green due to the formation of a cobalt-PAN complex. Then ethanol (7 μl) and chloroform (5 μl) were added. Afterwards, the sample was vortexed for 1 minute and subsequently left to reach phase separation. After centrifugation (10 g, 3 min, RT) spectrophotometric measurements were performed at 577 nm on Nanodrop showing that 2 μl of chloroform containing 92% of cobalt/PAN complex had passed into the second chamber 12.
Example 3
[0066] A mouse liver fragment (10 mg) was added to 1 ml of a phenol/chloroform/isoamyl alcohol solution (25:24:1 v/v/v; Merck) supplemented with the lipophilic dye Nile Red (Sigma, 200 μg/ml). The sample was then homogenized by a Pelletpestle® glass homogenizer (glasspestle microhomogenizer Pelletpestle®, Kontes) for 1 minute at 0° C. The lysate was transferred into a device constructed according to FIG. 2. The bottom of a conical polypropylene 1.5 ml Eppendorf tube (first chamber 21a) was perforated with six apertures 23a each with a diameter of 100 μm, and the bottom of second first chamber 21b was provided with one hydrophobized aperture 23b prepared as described in Example 2. The first chamber 21a was inserted into the first chamber 21b. These two first chambers 21a and 21b were placed above the second chamber 22 with a ventilation opening 24. The device was centrifuged for 5 min at 100 g and at 0° C.
[0067] After centrifugation, the inspection revealed that the second chamber 22 contained an intensely red colored chloroform phase (lipid and non-degraded RNA were not measured due to interference with Nile red). The first chamber 21b, provided with a hydrophobized aperture 23b, contained an aqueous phase (the total amount of DNA in this phase measured spectrophotometrically was 1 μg). The protein precipitate at the bottom and in the apertures of the first chamber 21a was analyzed (total of 95 μg, measured after dissolving the precipitate in a buffer containing sodium dodecyl sulfate by the BCA kit, Pierce).
Example 4
[0068] A blood sample was taken from a healthy volunteer into Vacutainer 4 ml Li-Hep tube and washed twice with 20 ml of PBS (2000 g, 10 min, RT). Then, an equal volume of PBS containing 100 mM sodium bisulfite and 100 mM dithionite was added to the blood cell column. The embodiment of the device according to FIG. 2, wherein the bottom of the first chamber 21 represented by the polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture 23a with external dimensions of 20×2 μm. The formed capillary aperture was then hydrophilized with a layer of dopamine and polyethyleneimine (PEI, J. Mater. Chem. A, 2 (2014) 10225-10230). Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) were dissolved in buffer containing Tris(hydroxymethyl)aminomethane (pH=8.5, 50 mM) both in a concentration of 2 mg/ml. Hydrophilization of the surface of the aperture 21a was performed with 1 ml of the prepared solution by pressure perfusion of the capillary aperture. After drying (24 h, at RT), the bottom of the first chamber 21a was covered with Parafilm and 50 μl of a red blood cell suspension was added into the first chamber 21a. Then, the sample was exposed to a carbon monoxide atmosphere for 60 minutes at room temperature using a COgen system (Sigma-Aldrich Product No. 744077). The cells were then lysed by adding 5 μl of 20% (w/w) solution of Triton X100 detergent in PBS. Measurement of a 5 μl aliquot of blood cells in a Nanodrop spectrophotometer at 420 and 432 nm (Clin. Chem. 30/6 (1984) 871-874) showed that exposure to CO gas caused the conversion of 98% hemoglobin in the lysate to carbonyl hemoglobin (COHb). Subsequently, the embodiment of the device included a cascade of three inserted PCRtubes (21a, 21b and 22)—the upper first chamber 21a contained a lysate of red blood cells (Parafilm foil was removed), the lower first chamber 21b (perforated at the bottom with the aperture 23b) contained a 120 μl column of DEAE sorbent. Sephadex A-50 (Sigma-Aldrich, product GE17-0180-02) was equilibrated in 0.01 M sodium phosphate buffer pH 7.5 by five times repeated centrifugation at 1000 g for 3 min at 20° C. and adding 50 μl of equilibration solution before each centrifugation. The non-perforated second chamber 22 as a collection chamber. This system was centrifuged at 1000 g, for 3 min, at 4° C. The premise and purpose of this arrangement was that the hemoglobin present in the blood cell lysate would be cleared of contaminating hydrophobic parts of the sample (hydrophobic parts of membranes, membrane proteins) remaining in the first chamber 21a (verified by SDS-PAGE), and most non-hemoglobin proteins of the lysate remained bound to the DEAE-Sephadex A-50 sorbent present in the middle tube 21b (Analytical Biochemistry 137 (1984) 481-484). This assumption was verified by non-denaturing electrophoretic analysis of proteins present in aliquots of the solution taken from the first chamber 21a and the second chamber 22, revealing that only hemoglobin was detected in the second chamber 22.
Example 5
[0069] A blood sample was taken from a healthy volunteer into Vacutainer 4 ml Li-Hep tube and washed twice with 20 ml PBS (2000 g, 10 min, RT). Then, an equal volume of PBS containing 100 mM sodium bisulfite and 100 mM dithionite was added to the blood cell column.
[0070] The embodiment of the device according to FIG. 1, where the bottom of the first chamber 11 formed by a polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture 13a with external dimensions of 20×2 μm. The prepared capillary aperture 13a was then hydrophobized by silanization (pressure perfusion of the capillary aperture with a 100 μl solution of dimethyldichlorosilane in 1,1,1-trichloroethane). After drying (24 h, at RT), the bottom of the first chamber was sealed with Parafilm foil and 50 μl of red blood cell suspension was pipetted into the first chamber. The blood cells were then lysed by adding 25 μl of 95% ethanol and 30 μl of chloroform while shaking the device (with the first chamber 11 covered with Parafilm foil) on a shaker (VortexGenie.2 mixer) 2 min at RT. This method of red blood cells lysis caused selective denaturation of hemoglobin, the most abundant protein present in the lysate (Chemosphere 88 (2012) 255-259). After removing the Parafilm foil and centrifuging the device at 100 g at 4° C., we verified by non-denaturing electrophoresis that only non-hemoglobin proteins were retained in the first chamber 11 (present in the aqueous phase above the denatured hemoglobin precipitate layer). In the second chamber 12 we observed approximately 25 μl of chloroform phase, where it is possible to further analyze the extracted lipids of blood cell membranes.
Example 6
[0071] The Example 6 describes separation in a system using dispersive liquid-liquid microextraction (with lighter organic solvents than water).
[0072] The embodiment of the device according to FIG. 1, wherein the bottom of the first chamber 11 formed by the polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture 13a with external dimensions of 20×2 μm. The prepared capillary aperture was then hydrophilized with a layer of dopamine and polyethyleneimine ((PEI), J. Mater. Chem. A, 2 (2014) 10225-10230). Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) were dissolved in a buffer containing Tris (hydroxymethyl) aminomethane (pH=8.5, 50 mM), both at a concentration of 2 mg/ml. Hydrophilization of the surface of the aperture 13a with this solution was performed by pressure perfusion of the capillary aperture with a volume of 1 ml. After drying (24 h, at RT), the bottom of the first chamber 11 was covered with Parafilm foil and 160 μl of a solution containing phenol (10 μg/ml, product 35952 Sigma-Aldrich) in MilliQ-deionized water was pipetted into the first chamber 11. 5 μl of extractant represented by 1-octanol (product 95446, Sigma-Aldrich) was then added. The first chamber was closed with a lid and shaken (VortexGenie.2 mixer) for 60 seconds at RT. Subsequently, the Parafilm foil was removed from the device and the device was centrifuged in a swinging rotor at RT for 3 minutes at 100 g. The analysis of first and second chamber content showed that the second chamber captured 160 μl of MilliQ-deionized water, while the 5 μl of 1-octanol remained in the first chamber. After reaction with ferric chloride, the 1-octanol phase was spectrophotometrically measured at 540 nm with a Nanodrop spectrophotometer. The result, based on the concentration curve of the absorbance of the phenol complex with iron ions, showed that the 1-octanol phase contained 1.6 μg of phenol.
Example 7
[0073] Microextraction Using a Solidified Organic Drop Microextraction (SFODME).
[0074] The embodiment of the device according to FIG. 1, where the bottom of the first chamber 11 formed by the polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture 13a with external dimensions of 20×2 μm. The prepared capillary aperture was then hydrophilized with a layer of dopamine and polyethyleneimine ((PEI), J. Mater. Chem. A, 2 (2014) 10225-10230). Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) were dissolved in a buffer containing Tris (hydroxymethyl) aminomethane (pH=8.5, 50 mM), both at a concentration of 2 mg/ml. Hydrophilization of the surface of the aperture 13a with this solution was performed by pressure perfusion of the capillary aperture with a volume of 1 ml and covered with Parafilm foil. Then 160 μl of ammonium metavanadate solution (1 mM NH.sub.4VO.sub.3, product 398128, Sigma-Aldrich) dissolved in aqueous sodium chloride solution (10 mM, pH 7) was added, followed by 5 μl of 8-hydroxychonoline solution (7 mM, product 252565 Sigma-Aldrich) in undecan-1-ol (product U1001 Sigma-Aldrich). The first chamber 11 was closed with a lid and shaken (VortexGenie.2 mixer) for 60 seconds at RT. Subsequently, the Parafilm foil was removed from the device, which was then cooled on ice and centrifuged at 100 g, for 3 minutes, at 4° C. in a swinging rotor. The inspection revealed that 160 μl of water passed into the second chamber 12, while 5 μl of the solidified phase of undecan-1-ol with extracted hydroxyquinoline-vanadium complex remained quantitatively in the first chamber 11. The contents of the first chamber 11 were warmed to room temperature, dissolved in 10 μl ethanol and measured at 383 nm on a Nanodrop spectrophotometer. Comparison of the absorbance of this sample with the concentration curve of the vanadium-hydroxyquinoline complex confirmed that approximately 8 μg of vanadium extracted from the aqueous solution into undecan-1-ol was present in the first chamber 11.
Example 8
[0075] A device corresponding to FIG. 2 was used to analyze the lipid component from a sample of exosomes and extracellular vesicles. The upper first chamber 21a was perforated with a 20×2 m hole and filled with 1 ml of Sephacryl S200 in PBS solution (pH 7.4). The aperture of the lower first chamber 21b was hydrophobized by silanization (pressure perfusion of a capillary aperture with 100 μl of a solution containing 2% dimethyldichlorosilane in 1,1,1-trichloroethane). After drying (24 h, at RT), the aperture in chamber 21b was covered with Parafilm foil and the chamber 21b was filled with 100 μl of chloroform. A 100 μl sample of blood plasma was loaded into the first chamber 21a and the system was centrifuged at 1000×g, for 5 min at 4° C. Subsequently the upper first chamber 21a was removed and the lower first chamber 21b was closed with a lid. The system was shaken (VortexGenie.2 mixer) for 60 seconds at room temperature and the Parafilm foil was removed from the first chamber. After stabilization of the phases, the system was centrifuged at 100×g, for 5 min at 4° C. The supernatant in the second chamber 22 containing the chloroform fraction enriched in the lipid component of the samples was analyzed on an API4000 tandem mass spectrometer (AB SCIEX) with pre-separation on an Agilent HPLC 1290 series liquid chromatography (Agilent). Free cholesterol (75%) was highest, followed by sphingomyelin (8%) followed by free cholesterol esters, ceramide monohexosides, monosial gangliosides and globotetraosylceramide.
Example 9
[0076] For gene therapy it is necessary to separate the vector contained in the plasmid DNA from the contaminating RNA. For this purpose, a device was prepared according to FIG. 1, wherein the bottom of the polypropylene PCR microtube PCR-02-C, 200 μl, Axygen was perforated with an aperture with external dimensions of 20×2 μm and surface treated by hydrophilization with a layer of dopamine and polyethyleneimine. Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) at a concentration of 2 mg/ml were dissolved in a buffer containing Tris (hydroxymethyl) aminomethane (pH=8.5, 50 mM). Hydrophilization of the aperture surface with this solution was performed by pressure perfusion of 1 ml of the solution. After drying (24 h, at RT), the aperture was covered with a layer of Parafilm foil and the embodiment of the device was set according to FIG. 1. A pooled sample of nucleic acids containing plasmid DNA and residual RNA was prepared from a bacterial lysate of the E. coli expression vector by a standard isolation method based on phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v; Merck) followed by precipitation of the nucleic acids in ethanol. 10 μl of the nucleic acid mixture was added to 90 μl of 10 mM Tris-HCl buffer pH 8 and 100 μl of the mixture containing 40 mM methyltrioctylammonium chloride, 250 mM lithium chloride and 0.5% (v/v) ethylhexanol in isooctane. The prepared sample was applied to the first chamber 11 of the device and left for 30 minutes at room temperature with gentle shaking. To accelerate the phase separation, the system was centrifuged at 2000 g, for 5 min, at RT. The Parafilm foil was removed from the first chamber 11 and the device was centrifuged at 100 g for 3 minutes in a swinging rotor. Subsequently, the aqueous phase of the sample located in the second chamber 12 was analyzed. By spectrophotometric analysis on Nanodrop, agarose gel, and using the RNAse assay, it was found that the aqueous phase contained only plasmid DNA.
Example 10
[0077] The embodiment of the device according to FIG. 1, wherein the bottom of the first chamber 11 formed by the polypropylene PCR microtube PCR-02-C, 200 μl, Axygen, was perforated with an aperture 13a with external dimensions of 20×2 μm. The prepared capillary aperture was then hydrophilized with a layer of dopamine and polyethyleneimine ((PEI), J. Mater. Chem. A, 2 (2014) 10225-10230). Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) were dissolved in a buffer containing Tris (hydroxymethyl) aminomethane (pH=8.5, 50 mM), both at a concentration of 2 mg/ml. Hydrophilization of the surface of the aperture 13a with this solution was performed by pressure perfusion of the capillary aperture with a volume of 1 ml of the solution. After drying (24 h, at RT), the aperture was covered with Parafilm foil and the device was assembled. A solution containing 29% (w/w) PEG 1000, 9% (w/w) PBS and 10% β-phycoerythrin (Merck, P1286) was applied to the first chamber 11 of the device. After stirring for 10 minutes at RT, the device was centrifuged at 1500 g for 10 minutes. The Parafilm foil was then removed and the device was centrifuged at 100 g for 3 min in a swinging rotor. Subsequently the PEG phase from the first chamber of the device 11 and the aqueous phase from the second chamber of the device 12 were analyzed by a spectrophotometer at wavelengths of 545 nm and 280 nm. From the absorbance ratio Abs.sub.545 nm/Abs.sub.280 nm, it was calculated that the PEG phase contained 77% of β-phycoerythrin.
Example 11
[0078] The bottom of Costar V-bottom polypropylene 96 well plate (Corning, N.Y., USA) was perforated at nine random wells 41 by apertures with external dimensions of 20×2 μm. The prepared capillary apertures were then hydrophobized by silanization (pressure perfusion of the capillary aperture with 100 μl solution of dimethyldichlorosilane in 1,1,1-trichloroethane). After drying (24 h, at RT), the bottom of the thus prepared first chambers was pressed firmly against rubber sheet and 10 μl of a solution containing liposoluble dye Sudan B (Sigma, 0.1 mg/ml) in chloroform and 170 μl of PBS (saline, phosphate buffered saline, 140 mM NaCl, 10 mM HEPES, pH 7.4) was added. The well plate of first chambers was sealed and vortexed for 1 min. Then the first chambers were inserted into the second chambers 42 represented by Costar V-bottom polypropylene 96 well plate. The assembled device was centrifuged at room temperature for 3 minutes at 100×g in a swinging rotor. The inspection revealed that 5 μl of Sudan B solution in chloroform had passed into the second chambers 42 and the colorless aqueous phase remained quantitatively in the first chambers 41. Spectrophotometric measurements at 600 nm on Nanodrop confirmed that the chloroform phase in the second chamber contained 98% of Sudan B.
Example 12
[0079] In order to evaluate the purification of glucose-6-phosphate dehydrogenase (G6PDH) produced by S. cerevisiae, the device according to FIG. 1 was used, wherein the bottom of the first chamber 11 was formed by an Eppendorf tube perforated with an aperture 13a with external dimensions of 20×2 μm. The prepared capillary aperture was then hydrophilized with a layer of dopamine and polyethyleneimine ((PEI), J. Mater. Chem. A, 2 (2014) 10225-10230). Dopamine hydrochloride (Sigma-Aldrich H8502) and PEI (Sigma-Aldrich product 408719, Mw 600 Da) were dissolved in a buffer containing Tris (hydroxymethyl) aminomethane (pH=8.5, 50 mM), both at a concentration of 2 mg/ml. Hydrophilization of the surface of the aperture 13a with this solution was performed by pressure perfusion of the capillary aperture with a volume of 5 ml of the solution. After drying (24 h, at RT), the aperture was covered with Parafilm foil and the device was assembled. A solution containing 17.5% (w/w) PEG 400, 15% (w/w) PBS and 1 g of yeast homogenate from S. cerevisiae was applied to the first chamber 11 of the device. After stirring at 8 rpm for 20 minutes at 10° C. the Parafilm foil was removed and the device was centrifuged at 2500 g for 10 minutes at 10° C. in a swinging rotor. Subsequently the PEG phase from the first chamber of the device 11 and the aqueous phase from the second chamber of the device 12 were analyzed by a spectrophotometer at wavelength of 340 nm following the rate of NADH.sup.+. From the absorbance was calculated that the enzyme recovery reached 97.7%.
Example 13
[0080] The same arrangement as in examples 1-12, but the emulsion was kept in motion either by immersing into sonicated water bath (Branson Ultrasonics CPX Series) or by shaking on IKA KS 130 orbital shaker (800/min) instead of vortexing.
Example 14
[0081] The same arrangement as in examples 1, 2, 3, 5 and 8, but the hydrophobisation of aperture was achieved by embedding polytetrafluorethylene dispersion (No. 665800 Sigma-Aldrich) on the aperture surface instead of silanization.
INDUSTRIAL APPLICABILITY
[0082] The device uses the principle of a capillary aperture for the passage of a fraction of the separated system. The device may in some embodiments allow parallel processing of many samples. The device is particularly suitable for use within pre-separations and separations of liquid-liquid systems. The device allows the separation of very low volume samples, for example in the order of units of microliters to tens of nanoliters. The devices with a serial arrangement of chambers also allows complex multistage separations with a combination of separation methods, which may include chromatography or solid phase extraction (SPE) utilizing antibodies, affinity agents, hydrophobic agents, hydrophilic agents, ionic agents or chelating agents, magnetic components, or components based on imprinted polymers, or combinations thereof.
