Patent Application
20230113229
The present invention provides a device and method for separation of components of samples, particularly suitable for low volume samples.
Chromatographic separation and liquid-liquid separation are commonly used methods in preanalytical preseparations, and preparative analytical processes. For the purposes of microsynthesis, during the screening of chemical libraries, for the analysis of very small samples or for the extraction of analytes from complex samples, it is desirable to process the samples in lowest possible volumes.
Chromatographic columns are packed with sorbent, which is kept in the column by a porous or selectively permeable barrier such as a frit, filter or membrane. The barrier prevents an outflow of the sorbent from the column together with the elution liquid. Similarly, a barrier is utilized by any separation method involving the presence of a solid phase.
A porous barrier, such as a frit, filter or membrane, always represents a large sorption surface and considerable dead volume that can affect and irreversibly bind or sorb a significant portion of the treated or analyzed sample. That poses major problems especially for low volume samples in terms of large reduction of sample yield or bias of the analysis.
To perform chromatographic liquid separation it is required to apply pressure forces, which include the application of overpressure, vacuum (negative pressure) or centrifugal force. The centrifugal force is commonly applied in chromatographic separation by spin microcolumns.
The negative effects of the barrier (frit/filter/membrane) are significantly manifested in very rare low volume samples, where the dead volume may even outweigh the sample volume and the large sorption surface may bind a substantial portion of the sample by adsorption. Suppliers commonly offer equipment suitable for volumes starting from 10 μl, separated on sorbents with a diameter of 40 μm. No suitable devices are currently available for submicrolitre sample volumes.
In the preseparations and separations in the liquid-liquid systems such as liquid-phase microextraction (LPME), dispersive liquid-liquid microextraction (DLLME), hollow-fiber or membrane liquid-liquid microextractions, single-drop microextraction (SDME), solidification of floating organic droplet (SFOD), ultrasonic-, vortex-, microwave- and air-assisted DLLME small sample volumes and parallel sample processing present also a complication. Separation of immiscible phases in a current liquid-liquid systems are essentially manual and serial techniques precluding parallel processing of many low volume samples.
A document WO 01/07138 describes a device comprising a perforated bottom container for separating a liquid phase of a low volume sample from a solid phase, wherein the solid phase may be a chromatographic sorbent. However, no device for handling small-volume liquid-liquid systems has been invented.
The present invention provides a device and method for the separation of components of a sample, particularly suitable for pressure separation of immiscible or partially miscible liquid systems (i.e., liquid systems with limited miscibility), optionally in combination with other separation methods. The device contains at least one first chamber with a V- or U-shaped bottom, which is perforated by at least one aperture with a diameter within the range of 1 to 100 micrometers (μm), preferably 1 to 40 μm, and at least the surface of each aperture is hydrophilized or hydrophobized. The device further contains a second (lower or the lowest) chamber surrounding the outside (i.e., the external surface) of the bottom of the first (upper) chamber.
The aperture in the first (upper) chamber is located at the tip or at the lowest point of the V- or U-shaped bottom. In this case the device is then suitable for use in swing rotor centrifugation, or for applying overpressure or vacuum (negative pressure) as a pressure force to stimulate sample flow. In an alternative embodiment of the device, the aperture in the upper chamber is located in a different position than at the tip or lowest point of the V- or U-shaped bottom. This embodiment of the device is suitable for use in angular rotor centrifugation, and the aperture is located at a point on the surface of the first (upper) chamber where the pressure force is highest during angular rotor centrifugation. Both embodiments can be combined to provide devices with variable applicability as further described.
The apertures represent passages through the wall of the first chamber. The apertures can be described as being capillary apertures. The surface of the aperture is the surface of the passage through the wall of the first chamber. In a preferred embodiment, the first chamber comprises 1 to 20, preferably 1 to 10, apertures.
The physical dimensions of the aperture in the bottom of the first chamber result in capillary properties, so that surface tension or steric restraint allows the permeation of only one of the liquids from the system (or the permeation of elution liquid in the case of chromatography sorbent elution) when a force (e.g. within the range of 1 to 10,000 g) is applied. The aperture may have a homogeneous diameter (the same diameter along the entire length of the capillary aperture), or it may not have a homogeneous diameter (thus the diameter changes in different sections of the length of the aperture). For example, the diameter of the aperture may be conical with a V- or U-shaped outlet at the lowest point of the first chamber. In the case of an inhomogeneous aperture diameter, the disclosed range of diameter sizes of the aperture corresponds to the smallest aperture diameter. The bottom of the first chamber is surrounded by a second chamber so that the outlet of the aperture leads into the second chamber.
The aperture in the bottom of the first chamber can be manufactured by physical, chemical or mechanical perforation, including but not limited to such means as penetration by a sharp object (e.g. a needle), by thermoshock (induced e.g. by cooling with liquid nitrogen or by rapid heating), by etching, by radiation in combination with chemical etching, by focused ionizing beam (e.g. electron beam lithography technique). The aperture may also be manufactured during the process of producing the first chamber by inserting a mandrel into a mold for casting or injecting the material forming the first chamber, or by a method of additive manufacturing (e.g. 3D printing).
The volume of the first chamber may preferably be of up to 10 ml, more preferably up to 5 ml or up to 2 ml, even more preferably in the range of 0.1 to 1000 μl.
The first chamber may be closable e.g. by a lid, a membrane or a foil.
At least the surface of the apertures in the first chamber must be hydrophilized or hydrophobized. In a preferred embodiment, also the inner surface of the bottom of the first chamber is hydrophilized or hydrophobized, wherein the inner surface of the bottom means at least the area of the inner surface of the chamber which contains the said at least one aperture. In another embodiment also the inner wall of the first chamber is hydrophilized or hydrophobized.
In some embodiments, the hydrophilization or hydrophobization means herein a surface treatment of the surface to be hydrophilized or hydrophobized. In some embodiments, the hydrophilization or hydrophobization includes incorporation of an auxiliary material into the material forming the surface or the chamber in order to increase hydrophilicity or hydrophobicity, respectively. The surface properties of the aperture in the first chamber are essential for the separation properties of the device used in the liquid-liquid separation mode. A hydrophobic surface of the aperture retains in the first chamber the hydrophilic fraction of a solution to be separated or, vice versa, a hydrophilic surface of the aperture retains in the first chamber the hydrophobic fraction of a solution to be separated.
Increasing the hydrophobicity or hydrophilicity, respectively, of a material may involve applying a hydrophobic or hydrophilic (respectively) coating; or chemical or physical treatment of the material (e.g. by laser or plasma); or application of a fabric with hydrophobic or hydrophilic properties onto the surface of the aperture and/or onto the surface of the bottom and/or onto the inner surface of the first chamber. Hydrophobic or hydrophilic, respectively, coatings as well as materials suitable for forming such coatings are known and commercially available to those skilled in the art. Examples of possible modifications are:
Separation methods demanding switching of hydrophobic and hydrophilic surface properties can be performed by utilization of versatile materials (e.g. TiO2, ZnO, TiO2/poly(methyl methacrylate), TiO2—SiO2/polydimethylsiloxane, poly(N-isopropylacrylamide), dendron thiol 2-(11-mercaptoundecanamido)benzoic acid attached to film of gold, (16-mercapto)hexadecanoic acid (2-chlorophenyl)diphenylmethyl ester attached to film of silver). Hydrophilic and hydrophobic properties of such materials and compounds can be altered by electric potential, temperature, pH or UV irradiation.
If needed, surface modification may require a prior surface activation or adjustment. Surface activation may be performed by chemical modification (strong oxidizing compounds, hydrolysis, aminolysis), electrochemical modification or physical method (e.g. Piezobrush® PZ2, Plasmabrush® PB3). Surface adjustment may include incorporation of compounds, layers or films (e.g. gold, silver, ZnO, TiO2, dopamine, etc.) in order to modify hydrophobic or hydrophilic properties or to provide a surface suitable for further modification by addition of compounds, layers or films.
If needed, microstructures or hierarchical structures may be provided on the surface. Micro- or nano-roughness of the surface can be achieved e.g. by high power oxygen. Multiple level hierarchical structures include e.g. micropits, spikes or pillar-like microstructures covered with nanobumps structure. Microstructures and hierarchical structures critically influence the hydrophobic and/or hydrophilic properties of materials.
The material of the chambers is preferably plastic, in particular polycarbonate; polyolefins such as polyethylene (PE), polypropylene (PP); polystyrene (PS); polyvinyl chloride (PVC); fluorinated polymers, such as Teflon (polytetrafluorethylene—PTFE).
The chamber material preferably has elasticity in the range of 0.01 to 8.5 GPa (Young's elasticity modulus).
The first and the second chambers may be specially produced to form the device of the invention. Alternatively, the device may be assembled from commonly available components which can serve as the first and second chambers, wherein at least one aperture is manufactured in the component forming the first chamber, which is hydrophilized or hydrophobized. Such commonly available components are, for example, Eppendorf-type tubes.
The second chamber surrounds the bottom of the first chamber from the outside. The first chamber can be resealable to prevent contamination of samples from the environment or cross-contamination between samples during separation, as well as sample evaporation, which is very undesirable in the case of low volume samples.
In a preferred embodiment, at least one ventilation opening can be provided in the device in the first and/or second chamber. These ventilation openings prevent the formation of undesired vacuum and/or overpressure during separation. In the first chamber the ventilation opening may be present in the side wall or in the closure. In the second chamber the ventilation opening may be present in the side wall of the chamber.
In the device of the invention, the chambers are connected in such a way that at least the bottom of the first chamber is surrounded on the outside by the second chamber, so that the liquid flowing out of the aperture in the bottom of the first chamber flows into the second chamber. The chambers can also be connected in a gas-tight way, e.g. to create an overpressure which can define the volume of fluid flowing through the aperture.
In a preferred embodiment, the device may include a plurality of first chambers and a plurality of second chambers, which allows a plurality of separations to be performed in parallel and/or simultaneously. This arrangement is hereinafter referred to as a “parallel arrangement”. The first chambers may be arranged in one holder to form a first chamber system and the second chambers in another holder to form a second chamber system, and the first chamber system is inserted into the second chamber system. Optionally, multiwell plates placed on top of each other may be used, with apertures being formed in the bottoms of the wells of the first plate forming the first chambers, and the plates being then placed on top of each other so that the wells of the second plate surround the wells of the first plate from the outside.
For example, the multiwell plates may contain 6, 12, 24, 48, 96, 384, 1536, 3456 wells, but the number of the first and the corresponding second chambers may also be arbitrary. The dimensions and arrangement of the wells of commercially available multiwell plates are standardized and therefore suitable for automatic handling by existing automatized handling systems (e.g., lab robots) and software. For parallel separations it is advantageous to ensure that the apertures in the bottoms of all wells forming the first chambers are substantially identical.
In some embodiments, the device may also include a plurality of first chambers inserted into each other. Thus, when using N first chambers, (N−1) first chambers surround the bottom of the previous first chamber in the direction of liquid flow. This allows the sample to be applied to one first chamber, and upon application of pressure, the sample passes successively through all the other first chambers. The last first chamber in the direction of liquid flow is inserted into the second chamber, i.e. at least its bottom part is surrounded by the second chamber. This results in a gradual separation of the sample or a gradual sorption of various components of the sample and separation of various components of the liquid-liquid system in the various first chambers. This arrangement is hereinafter referred to as a “serial arrangement”.
At least one of the first chambers in the serial arrangement is the first chamber including the hydrophilized or hydrophobized surface of at least the aperture(s) as described above. Further first chambers may be chambers with a V- or U-shaped bottom, at least one aperture with a diameter in the range of 0.1 to 100 μm, preferably 1 to 40 μm, located at the V-tip or the lowest U-shaped point, with or without surface treatment in at least part of the inner surface.
The treatment in at least part of the inner surface includes the presence of separation means on at least the surface of the aperture, these separation means may be for example antibodies; affinity, hydrophobic, hydrophilic, ionic or chelating agents; magnetic components; or components based on imprinted polymers; optionally a combination of the aforementioned means and properties may be provided. The separation means bind specifically at least one component of the sample upon use of the device.
The separation means may be present on at least one capillary aperture, on the inner surface of the bottom of the first chamber or on the entire inner surface of the first chamber.
When the chamber does not contain any separation means, it is usually intended to fill them with a particulate sorbent, such as a sorbent suitable for solid phase extraction (SPE).
The particulate sorbent may be any sorbent suitable for sample separation. Examples of particulate sorbents include sorbents used for gel filtration, ion exchange, hydrophobic, affinity, or metal chelate affinity chromatography, or for the technique of molecular imprinted polymers; more specific examples of sorbents are listed in Table 1.
In some embodiments, the device may contain a plurality of first chambers and a plurality of second chambers, which are arranged in a parallel arrangement of a plurality of serial arrangements, wherein each serial arrangement contains a plurality of first chambers and one second chamber. Thus, the embodiments contain a matrix of second chambers, wherein each second chamber holds a column of first chambers inserted one into another.
Such embodiment enables parallel separation of a plurality of samples, wherein each sample is subjected to the same separation conditions and passes through the device at the same time. At least one of the first chambers in each serial arrangements contains the hydrophilized or hydrophobized aperture(s) as described above. The other first chambers may be as described herein above for the serial arrangement of the device.
The present invention further provides a method for separation of components of a sample in a liquid-liquid system performed in the device described herein above, comprising the following steps:
The pressure force here includes overpressure, vacuum (negative pressure), centrifugal force, or gravitational force. The pressure force acts on the system in the first chamber in the direction towards the aperture. The pressure force can be caused, for example, by overpressure in the first chamber, vacuum (negative pressure) in the next chamber (first or second), or by centrifugal or gravitational force acting on the whole device.
The pressure force acts towards the bottom or wall of the first chamber so that the sample is pushed towards the aperture in the bottom or wall of the first chamber, and the respective fraction of the sample (with the same hydrophilic/hydrophobic nature as the nature of the aperture and optionally its surrounding area) passes through this aperture into the second chamber. The pressure force can be applied as an overpressure from above, e.g. by means of a piston, as a vacuum (negative pressure) using a vacuum (low pressure) in the next or second chamber, or as a gravitational or centrifugal force when centrifuging the device, the centrifugal force acting towards the aperture in the bottom of the first chamber. Conventional laboratory centrifuges or microcentrifuges can be used in the centrifugation step.
The analyte can be of liquid, solid or gas nature. The sample may contain a liquid substance, a mixture of liquid substances, a liquid solution of solid substances, a liquid solution of a mixture of solid substances, a liquid solution of a liquid substance, a liquid solution of a mixture of liquid substances, or a liquid solution of a mixture of solid and liquid substances. Furthermore, analytes can be absorbed from gas phase into the liquid sample, or a gas sample may be introduced directly into the first chamber. Optionally, the fluid sample (i.e. liquid or gas sample) may be an eluent extracting analytes from a sorbent, or gas or liquid component(s) from a previous separation step.
When a serial arrangement of chambers of the device is used, the sample and the system of immiscible liquids are introduced into the topmost first chamber, and a pressure force is generated in order to make the sample pass sequentially through all the first chambers into the second chamber, in which the last fraction of the sample is retained. The first chambers without hydrophilic/hydrophobic modification are usually filled with a solid phase sorbent or have separating means provided at least on the surface of the aperture and/or onto the surface of the bottom and/or onto the inner surface of the first chamber (the separating means including e.g. antibodies, affinity, hydrophobic, hydrophilic, ionic or chelating agents, magnetic components, or components based on imprinted polymers, or combination thereof). The individual fractions (or components) of the sample are gradually separated in the first chambers by application of various separation means or a solid phase sorbent, while at least one first chamber causes the separation of a system of immiscible liquids based on hydrophilized or hydrophobized aperture surface.
The separation described above takes place simultaneously for all samples when a parallel arrangement of chambers or a parallel arrangement of serial arrangements of chambers is used.
The device of the invention has a construction which does not require the presence of a frit, filter or membrane, while still selectively separating components (or fractions) of samples based on (inter alia) hydrophobic and hydrophilic properties. The device of the invention enables flow of the sample and the liquid system in which the sample occurs through the device, wherein fractions of the sample are retained in the first chamber(s), until the last sample fraction flows into the second chamber. Such device, in comparison to similar available devices, eliminates complications such as the existence of dead volume, prevents disruption of the separation process and increases the yield of the separation. Additional advantages of the device in comparison to commercially available devices include simplification of the production due to the absence of frit, filter or membrane, simple production of devices allowing parallel separations of a plurality of samples (also in serial arrangement). The absence of a frit, filter or membrane further avoids irreversible binding of sample fractions and avoids interference with the separation process which are common in known devices. Thus the device of the invention enables processing of samples with volumes in the order of nanolitres, including parallel processing of tens to thousands of samples.
Examples of various embodiments of the device according to the invention are shown in
The bottom of the first chamber 11 of the device according to
The bottom of the first chamber 11 of the device according to
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
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).
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
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.
The embodiment of the device according to
The Example 6 describes separation in a system using dispersive liquid-liquid microextraction (with lighter organic solvents than water).
The embodiment of the device according to
Microextraction Using a Solidified Organic Drop Microextraction (SFODME).
The embodiment of the device according to
A device corresponding to
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
The embodiment of the device according to
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.
In order to evaluate the purification of glucose-6-phosphate dehydrogenase (G6PDH) produced by S. cerevisiae, the device according to
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.
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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| PUV2020-37201 | Jan 2020 | CZ | national |
| PV 2020-47 | Jan 2020 | CZ | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CZ2021/050011 | 1/28/2021 | WO |
