Method for Separation of Biopolymer Molecules and a Carrier for Application of this Method

Abstract

The invention relates to a method for separation of biopolymer molecules, particularly biopolymer molecules from the group consisting of mono- a multi-phosphorylated peptides, recombinant peptides/proteins with a polyhistidine tag (His-tag) or with another chemically similar biospecific tag, cysteine-containing peptides/proteins and nucleic acids, in which a biopolymer molecule is bound in a binding solution by a specific binding to a carrier, which contains a core with dimensions in nano- and/or submicro- and/or microscale, which is composed of oxide of at least one transition metal and/or silicon oxide, on whose surface is deposited at least one continuous or non-continuous layer and/or nanoparticles of magnetic metal oxide and/or such nanoparticles are deposited in its inner structure, and subsequently undesirable and non-specifically bound components are washed off at least once from the carrier-bound bio-molecules by a washing solution, whereupon biopolymer molecules are eluted from it by changing pH and/or by using an elution solution. The invention also relates to a carrier for application of this method.

Claims

1. A method for separation of biopolymer molecules, particularly biopolymer molecules from the group consisting of mono- and multi-phosphorylated peptides, recombinant peptides/proteins with a polyhistidine tag or another chemically similar biospecific tag, cysteine-containing peptides/proteins and nucleic acids, characterized in that a biopolymer molecule from the group consisting of mono- and multi-phosphorylated peptides, recombinant peptides/proteins with a polyhistidine tag or another chemically similar biospecific tag, cysteine-containing peptides/proteins, nucleic acids, is bound in a binding solution by a specific binding to a carrier which contains a core having dimensions in nano- and/or submicro- and/or microscale, formed by oxide of at least one transition metal, on whose surface is deposited at least one continuous or non-continuous layer and/or nanoparticles of magnetic metal oxide and/or such nanoparticles are deposited in its inner structure, and subsequently undesirable and non-specifically bound components are washed off at least once from the carrier-bound biomolecules by a washing solution, whereupon the biopolymer molecules are eluted from it by changing pH and/or by an elution solution.

2-26. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0048] FIG. 1 of the enclosed drawings shows a 100,000 times magnified photograph of a carrier developed for separation of biopolymer molecules according to the invention in one variant of embodiment,

[0049] FIG. 2 shows four flow charts of the method for isolation of four different biopolymer molecules according to the invention,

[0050] FIG. 3 shows three MS spectra comparing clean standard—phosvitin cleaved by trypsin, the result of the enrichment of phosvitin with the aid of a commercial carrier known from the background art and the result of the enrichment of phosvitin with the aid of the carrier according to the invention in FIG. 1,

[0051] FIG. 4 shows two MS spectra comparing standard—commercial ubiquitin with a His-tag cleaved by trypsin, the result of separation of ubiquitin using the carrier according to the invention in the alternative according to FIG. 1 and finally,

[0052] in FIG. 5 there are two MS spectra comparing standard—bovine serum albumin cleaved by trypsin, and the result of separation of peptides from bovine serum albumin by means of the carrier according to the invention in the alternative according to FIG. 1.

SPECIFIC DESCRIPTION

[0053] The method for separation of biopolymer molecules (particularly mono- and multi-phosphorylated peptides, recombinant peptides/proteins with a polyhistidine tag (His-tag) or another chemically similar biospecific tag, cysteine-containing peptides/proteins and nucleic acids) according to the invention is based on using a composite carrier composed of a core consisting of a material based on oxide of transition metal/metals (e.g. Ti, Al, Zr, Ta, Hf, W, Nb, Sn, V, Fe) or silicon oxide, on whose surface and/or in whose inner structure is deposited magnetic metal oxide (e.g. Fe, Co and Ni).

[0054] The core of this carrier is composed of substantially any formation, e.g. by a spherical or substantially spherical particle, a tube (a hollow elongated object, whose length is considerably greater than its diameter), a fiber (a solid elongated object, whose length is considerably greater than its diameter), or substantially any other object/particle (regular or Irregular, e.g., a cube, a rectangular parallelepiped, a prism, a pyramid, etc.), having dimensions in nano-, sub micro- or microscale. In particular, its material is transition metal oxide (preferably, e.g., TiO.sub.2, ZrO.sub.2, Al.sub.2O.sub.3, Ta.sub.2O.sub.5, WO.sub.3 SnO.sub.2, HfO.sub.2, Nb.sub.2O.sub.5, MoO.sub.2, MoO.sub.3, ZnO, V.sub.2O.sub.5, Fe.sub.2O.sub.3, Fe.sub.3O.sub.4, or a mixture of at least two of them in any ratios or proportions, or a mixture of both stoichiometric and nonstoichiometric forms of at least one of them in any ratios), silicon oxide (SiO.sub.2— in any ratios of stoichiometric and nonstoichiometric forms), or a mixture of at least one transition metal oxide and SiO.sub.2 in any ratios. Preferably, especially TiO.sub.2 can be used as a material of the core of the carrier as the most biocompatible of all the oxides which have been mentioned above.

[0055] On the surface of this core and/or in its inner structure is deposited magnetic metal oxide, preferably especially any stoichiometric or nonstoichiometric variants of Fe.sub.xO.sub.y, NiO, CoO, Co.sub.3O.sub.4, ferrite based on M.sub.2O.sub.3, where M is a metal from the group consisting of Fe. Ni, Co Fe, Ni, Co. or a mixture of at least two of them. Also, it is advantageous if we use magnetite and/or maghemite, which are not toxic for the human body and which exhibit—supposed they are in the form of particles smaller than 12 nm—superparamagnetic properties. Furthermore, this material can be deposited on the surface of the core in the form of continuous or noncontinuous layers and/or randomly dispersed nanoparticles, and/or is deposited in the form of nanoparticles in its inner structure, ideally in such a manner that at least part of some nanoparticles protrude onto the surface. In the first alternative, nanoparticles of magnetic metal oxide on the surface of the core are preferably tagged by covalent or other physico-chemical interactions which ensure maximum protection from external, especially physical influences (washing off, releasing, etc.), thus ensuring long-term dynamics of a particular carrier in the magnetic field.

[0056] The main advantage of the carrier according to the invention is the fact that it enables the specific surface interaction of the transition metal oxide with biopolymer molecules which are to be separated, while maintaining at the same time the contribution of its magnetic material which allows to effectively remove the carrier from the sample due to the action of the magnetic field; moreover, this carrier has a huge specific surface. Another benefit of the carrier according to the invention is its extensive chemical, physical and mechanical compatibility—in different variants there are in principle very similar materials which can be replaced with one another and/or combined.

[0057] The most advantageous variant of the carrier is the variant in which its core is composed of a TiO.sub.2 nanotube on whose surface are deposited Fe.sub.3O.sub.4 nanoparticles (FIG. 1), since these materials do not exhibit toxicity for the human organism, are biocompatible and, at the same time, they can be used repeatedly since they sustain high binding ability and selectivity.

[0058] In addition, the carrier according to the invention can be effectively decontaminated by using photocatalysis and sterilized by the application of UV light Irradiation.

[0059] During the production of the carrier according to the invention, magnetic metal oxide is preferably applied to pre-prepared cores, e.g. by a direct chemical method, using a method of sputtering, a method of atomic layer deposition (ALD—Atomic Layer Deposition).

[0060] The carrier thus obtained exhibits both sufficient mechanical and chemical resistance, and so it can be used for efficient separation of biopolymer molecules in which the biopolymer molecules to be separated are specifically bound to the carrier, whereupon undesired and non-specifically bound components from a particular sample are washed off, and finally the biopolymer molecules to be separated are eluted by changing pH or with a suitable solution. Biopolymer molecules separated by this process achieve thanks to this high purity.

[0061] Magnetic properties of the composite carrier according to the invention allow a variety of uses—whether it is batch or column arrangement in the form of a magnetically stabilized fluidized bed (Magnetically Stabilized Fluidized Bed—MSFB). Spherical carrier variants can be in a classical column arrangement, whereas fibrous arrangements can be used, for example, for the production of filters which will specifically isolate the target substances. In all these cases, a strong magnetic field is used for the purpose of stabilization of the separation bed and/or efficient extraction of carriers with bound biopolymer molecules from the reaction mixture.

[0062] Preferably, the magnetic material deposited on the surface of the core of the carrier and/or in its inner structure has superparamagnetic properties, due to which the carrier after removal of the magnetic field does not exhibit residual magnetism (which ferromagnetic materials would exhibit), and so it is possible to work with it alternately in the mode of homogeneously dispersed suspension or in separate phases.

[0063] In another alternative of separation of biopolymer molecules, the carrier that has been described above is incorporated into separation channels of microfluidic systems (the so-called μTAS), by which means the positive effect of miniaturization of reaction volume during proof or separation of a particular biopolymer molecule is multiplied, although the specific surface area is still high due to the nature of the filling of the separation microchannel of the system.

[0064] Another alternative of performing the method for separation of biopolymer molecules is capturing a compact block of carriers, whose core is composed of flowthrough nanotubes (their lower closed portion formed during the production being removed), in a holder of a membrane, whereby the mixed solution of the substances flows through the cavities of the nanotubes in a similar manner as in the case of membrane filtration processes.

Separation of Mono- and Multi-Phosphorylated Peptides

[0065] In the initial medium—e.g. in a mixture of peptides/proteins, cell lysate, cell growth medium, etc., the proteins contained are first cleaved into shorter peptide chains. Subsequently, the mono- and multi-phosphorylated peptides thus obtained are bound by a specific interaction between their phosphate group and metal oxide to the carrier according to the invention. The actual process of the binding of the peptides to the carrier takes place in a binding solution which contains 35-90% of organic phase (preferably acetonitrile (ACN), methanol, ethanol or another organic solvent) and 0.1-5% of carboxylic acid (preferably trifluoroacetic acid (TFA), formic acid, etc.), whereby low pH of this solution is used to suppress the non-specific binding. In addition, to reduce the non-specific binding, it is possible to use a different carboxylic acid, e.g. lactic acid (preferably with a concentration of 0.5 to 2 M), glycolic add, salicylic acid, phthalic acid, 2,5-dihydroxybenzoic acid (preferably with a concentration of 0.5 to 3 M), etc.

[0066] After the binding, undesirable and non-specifically bound components from the carrier are washed off at least once with a washing solution which contains from 5 to 90% of organic phase (preferably ACN, methanol, ethanol or another organic solvent) and 0.1-5% of carboxylic acid (preferably TFA, formic acid, etc.), or with a binding solution.

[0067] After that, specifically bound mono- and/or multi-phosphorylated peptides are released from it with an elution solution with a pH higher than 9 (e.g. 1% aqueous ammonia solution). In another variant, elution can be carried out competitively, using a solution containing phosphate/phosphates (at least 20 mM).

[0068] Binding, washing and elution steps are assisted by a magnetic field, when the carrier is separated from the liquid.

[0069] The separated mono- and multi-phosphorylated peptides can be further used, e.g. for subsequent (MS) analysis.

[0070] After the separation, the carrier of the Invention in case of need is regenerated (see below).

[0071] Separation of mono- and multi-phosphorylated peptides is indicated in FIG. 2 in the first flow chart from the left-hand side.

Example 1

[0072] 1 mg of the carrier according to the invention, which was previously stored in a solution containing 80% of acetonitrile (ACN) and 0.1% of trifluoroacetic acid (TFA), was washed under the influence of a magnetic field with 500 μl of a washing solution containing 80% of ACN and 0.1% of TFA, and after that with 500 μl of the binding and washing solution containing 80% of ACN, 5% of TFA and 1 M of lactic acid (LA). The excess solution was removed and to the carrier was added 200 μl of a solution containing 60 pmol of proteolytically cleaved phosvitin—model hyperphosphorylated protein, dissolved in a binding and washing solution having the composition as described above. The carrier with applied cleaved phosvitin was afterwards incubated under gentle rotation at room temperature for 60 min.

[0073] After incubation, the binding and washing solution containing unbound components was removed from the reaction mixture and the carrier was washed with 2×500 μl of a washing solution containing 80% of ACN, 5% of TFA and 1 M of LA and with 2×500 μl of a washing solution containing 80% of ACN and 0.1% of TFA. 50 μl of an elution solution (1% aqueous ammonia solution) was then added to the carrier thus washed, whereby elution proceeded under rotation for 15 minutes.

[0074] Then, after the elution solution above the carrier was removed, it was acidified with TFA and analyzed by means of mass spectrometry (MS).

[0075] FIG. 3 shows three MS spectra—at the top there is a MS spectrum of a clean standard—phosvitin, in the middle there is a MS spectrum of phosvitin separated by using the most commonly used type of a carrier—particularly Titansphere™ TiO Bulk Material, 10 μm produced by GL Sciences Inc., and at the bottom there is a MS spectrum of phosvitin separated by using the carrier according to the invention in the variant according to FIG. 1, when its core is composed of TiO.sub.2 nanotubes with Fe.sub.3O.sub.4 nanoparticles deposited on their surface. The comparison of these spectra clearly demonstrates that the ratio between the multi-phosphorylated peptides—peaks 1620 and 1149, and the mono-phosphorylated peptides—peaks 1540 and 1069, is when using the carrier according to the invention shifted more towards the multi-phosphorylated peptides. Also, peaks 1540 and 1620, as well as 1149 and 1069, only differ in the amount of phosphogroups and have the same amino acid sequence, which means that the carrier according to the invention separates the multi-phosphorylated peptides better than the carrier Titansphere™ TiO. Moreover, a purer sample is obtained under the same conditions, and when using the carrier according to the invention, MS spectrum has one order higher Intensity, which indicates high capture efficiency, as well as high selectivity.

[0076] Owing to the fact that separation according to the invention is based on the specific interaction between a phosphate group mono- or multi-phosphorylated peptide and the metal oxide of the carrier according to the invention, it is apparent that when using the same or similar technique of separation, the other mono- a multi-phosphorylated peptides will behave in the same or similar manner as above-mentioned phosvitin.

Separation of Peptides/Proteins Containing His-Tag

[0077] In the initial medium—e.g. a mixture of peptides/proteins, cell lysate, cell growth medium, etc., for separation at the peptide level at first the contained proteins are proteolytically cleaved into shorter polypeptide chains. For separation at the protein level, the proteins are separated in an intact form. In both cases, the carrier according to the invention is used, whereby separation of peptides/proteins is based on the specific Interaction between positively charged groups around atom N in the structure of histidine with oxide of metal/metals of the carrier.

[0078] Preferably, binding of peptides to the carrier occurs in a binding solution containing 10 mM of imidazole in 100 mM of glycin-HCl buffer with pH 3.2, whereby the same solution is then preferably used also for washing off undesirable and non-specifically bound components.

[0079] In the case of proteins, for binding to the carrier and for washing off undesirable and non-specifically bound components from the carrier, a solution consisting of 50 mM of Tris-HCl buffer with a pH of 7.5 is preferably used.

[0080] In general, it is possible to use a binding and/or washing solution which contains a buffer of any composition in the pH range of 3 to 8.7, with the addition of 0-300 mM of imidazole. Generally, it is possible to use any buffer or any substance which has the given range of pH and at the same time is not an elution agent. For the purpose of suppressing non-specific binding, the solutions can be further supplemented with salts, for example NaCl, up to a concentration of 0.5 M.

[0081] Peptides are eluted from the carrier by changing pH in the range between 9.5 and 12 or with elution solution/solutions containing a minimum of 20 mM of phosphate/phosphates (for example, disodium phosphate or a phosphate buffer). Proteins are eluted from the carrier by additional imidazole or increasing its concentration compared to the binding and washing solution (to 10 mM-1 M) or also with elution solution/solutions containing a minimum of 20 mM of phosphate/phosphates (for example, disodium phosphate or a phosphate buffer)

[0082] If for some reason it is desirable only to remove peptides from the solution, it is possible to leave them bound to the carrier and eliminate them afterwards during its regeneration. This procedure is applicable, e.g., for removing the cleaved off polyhistidine tags.

[0083] The binding, washing and elution steps are assisted by the magnetic field by which the carrier is being separated from the liquid.

[0084] After the separation the carrier according to the invention in case of need is regenerated (see below).

[0085] Separation of peptides/proteins containing His-tag is indicated in FIG. 2 by the second flow chart from the left-hand side.

Example 2—Separation of Peptides Containing His-Tag

[0086] 1 mg of the carrier according to the invention, which was previously stored in a solution containing 80% of ACN and 0.1% of TFA, was washed under the influence of the magnetic field with 2×500 μl of a washing solution consisting of 0.1 M of glycin-HCl buffer with pH 3.2. To the washed carrier was then added 60 pmoles of proteolytically cleaved ubiquitin with a His-tag dissolved in 200 μl of a binding solution composed of 0.1 M of glycine-HCl buffer with pH 3.2. The carrier with applied ubiquitin was then incubated under gentle rotation at room temperature for 60 min.

[0087] After incubation, the binding solution with unbound components was removed from the test tube and the carrier was washed with 2×300 μl of a washing solution consisting of 0.1 M of glycin-HCl buffer with pH 3.2, with 2×300 μl of a washing solution containing 0.1 M of glycin-HCl buffer with pH 3.2 and 150 mM of imidazole, and after that with 300 μl of a washing solution consisting of 0.1 M of glycin-HCl buffer with pH 3.2.

[0088] 100 μl of an elution solution (1% aqueous ammonia solution) was added to the carrier thus washed, whereby elution proceeded under rotation for 15 minutes. After the elution was completed, the elution solution above the carrier was removed, the solution was acidified with TFA and analyzed by means of MALDI-MS.

[0089] FIG. 4 shows 2 MS spectra—at the top there is a MS spectrum of commercial His-tagged ubiquitin cleaved by trypsin, the lower one is a MS spectrum of ubiquitin separated with the aid of the carrier according to the invention in the alternative according to FIG. 1, when its core is formed by TiO.sub.2 nanotubes with Fe.sub.3O.sub.4 nanoparticles deposited on their surface. It follows from the comparison of the two spectra that during separation almost pure His-tagged ubiquitin was obtained (peak 1768.840) with amino acid sequence GSSHHHHHHSSGLVPR. This confirms high affinity of the carrier according to the invention to recombinant peptides/proteins with a polyhistidine tag.

Example 3—Separation of His-Tagged Proteins

[0090] 5 mg of the carrier according to the invention, which was previously stored in a solution containing 80% of ACN and 0.1% of TFA, was washed under the influence of the magnetic field with 2×500 μl of a washing solution consisting of 50 mM of Tris-HCl buffer with pH 7.5. To the washed carrier was then added 200 μl of a binding solution composed of 50 mM of Tris-HCl buffer with pH 7.5, which contained recombinant His-tagged protein. The carrier with the applied proteins was afterwards incubated under gentle rotation at room temperature for 60 min.

[0091] After incubation, the binding solution with unbound components was removed from the test tube and the carrier was washed with 5×500 μl of a washing solution consisting of 50 mM of Tris-HCl buffer with pH 7.5.

[0092] After that, 2×100 μl of an elution solution containing 50 mM of Tris-HCl buffer with pH 7.5 and 150 mM of imidazole were added to the carrier thus washed, whereby elution proceeded under rotation for a period of 2×15 minutes. After the completion of the elution process, the elution solution above the carrier was removed and both the elution and washing solution were analyzed by means of polyacrylamide gel electrophoresis in sodium dodecyl sulfate environment, whereby it was found that the electrophoretogram of the elution fraction, unlike the original sample, contains pure recombinant protein with minimal impurities.

[0093] With regard to the fact that separation according to the invention is based on a specific interaction between the positively charged group around atom N in the structure of histidine with the metal oxide of the carrier according to the invention, it is apparent that the other peptides/proteins with a His-tag or another chemically similar biospecific tag, will behave in the same or similar manner when using the same or similar technique of separation.

Separation of Cysteine-Containing Peptides/Proteins

[0094] In the initial medium—e.g. a mixture of peptides/proteins, cell lysate, cell growth medium, etc., for the separation at the peptide level, at first the proteins contained are proteolytically cleaved into shorter polypeptide chains. For separation at the protein level, the proteins are separated in an intact form. Peptides or proteins with cysteines are then separated using the carrier according to the invention on the basis of the specific interaction between —SH group of cysteine and the metal oxide of the carrier. The free —SH group, if it is no longer present, is obtained by the preceding reduction of —S—S— bonds by means of dithiotreitole (at least 10 mM).

[0095] Similarly, cysteine-containing peptides/proteins can be isolated by means of the specific interaction between —SO.sub.3H group and the metal oxide of the carrier. The free —SO.sub.3H group is obtained by a preceding oxidation of —SH group or directly from —S—S— bonds by means of performic acid (at least 2%).

[0096] Binding of peptides/proteins to the carrier then takes place in a binding solution containing 100 mM of Tris-HCl buffer with pH 7.5 and 1% of sodium dodecyl sulfate (SDS). In general, it is possible to use a binding solution which contains a buffer of any composition with a pH in the range between 6 and 8 with additional 0-2% of SDS. Generally, it is possible to use any buffer or any substance which has the given range of pH and at the same time is not an elution agent. The same solution is then used also as a washing solution for washing off undesirable and non-specifically bound components from the carrier. For the purpose of suppressing non-specific binding, the binding and washing solution can be further enriched with 10-50 mM of dithiothreitol, mercaptoethanol or mercaptobenzoic acid.

[0097] Afterwards, elution is carried out by increasing pH to a value from 10 to 12 or with an elution solution containing at least 20 mM of phosphate/phosphates.

[0098] The binding, washing and elution steps are assisted by the magnetic field by which the carrier is being separated from the liquid.

[0099] After the separation the carrier according to the invention in case of need is regenerated (see below).

[0100] Separation of peptides/proteins containing cysteine is indicated in FIG. 2 by the second flow chart from the right-hand side.

Example 4

[0101] Separation of peptides containing cysteine/cysteines with a free —SH group from a model sample—bovine serum albumin cleaved by trypsin—was carried out with the aid of the carrier according to the invention. 0.5 mg of this carrier, which was previously stored in a solution containing 80% of ACN and 0.1% of TFA, was washed under the influence of the magnetic field with 2×500 μl of a washing solution containing 100 mM of Tris-HCl buffer with pH 7.5 and 1% of sodium dodecyl sulfate (SDS). To the washed carrier was then added a binding solution with 60 pmol of a model sample in 200 μl 100 mM of Tris-HCl buffer with pH 7.5, with additional 1% of SDS. The carrier with the applied sample was afterwards incubated under gentle rotation at room temperature for 60 min.

[0102] After incubation, the binding solution with unbound components was removed from the test tube and the carrier was washed with 6×300 μl of a washing solution containing 100 mM of Tris-HCl buffer with pH 7.5 and 1% of SDS.

[0103] After that, 100 μl of an elution solution (1% aqueous ammonia solution) was added to the washed carrier, whereby elution proceeded under rotation for a period of 15 min. After the completion of the elution, the elution solution above the carrier was removed and both the elution and washing solution were analyzed by means of mass spectrometry (MS).

[0104] FIG. 5 shows 2 MS spectra—the upper one is a MS spectrum of the standard, the lower one is a MS spectrum of peptides separated with the use of the carrier according to the invention in the variant according to FIG. 1, when its core is formed by TiO.sub.2 nanotubes with Fe.sub.3O.sub.4 nanoparticles deposited on their surface. Moreover, cysteine-containing peptides are marked with arrows, whereby, e.g., peak with the highest intensity in the lower MS spectrum (1052.450) with amino acid sequence CCTKPESER is in the upper spectrum completely invisible. That demonstrates the selectivity of the carrier according to the invention preferentially for cysteine-containing peptides.

Example 5

[0105] 1 mg of a carrier according to the invention, which was previously stored in a solution containing 80% of ACN and 0.1% of TFA was under the influence of a magnetic field washed with 2×500 μl of a washing solution containing 100 mM of Tris-HCl buffer with pH 7.5 with additional 1% of SDS. To the washed carrier was added a mixture of proteins (60 pmol of each) containing bovine serum albumin, in which —S—S— bonds (disulfide bridges), were reduced by means of 20 mM of dithiothreitol.

[0106] This mixture was added to the carrier in 200 μl of a binding solution containing 100 mM of Tris-HCl buffer with pH 7.5 with additional 1% of SDS. The carrier with the applied proteins was afterwards incubated under gentle rotation at room temperature for 60 min.

[0107] After incubation, the binding solution with unbound components was removed from the test tube and the carrier was washed with 6×300 μl of a washing solution containing 100 mM of Tris-HCl buffer with pH 7.5 with additional 1% of SDS.

[0108] 100 μl of an elution solution (1% aqueous ammonia solution) was added to the carrier thus washed, whereby elution proceeded with rotation for a period of 15 min.

[0109] After the completion of the elution, the elution solution above the carrier was removed and this solution was acidified (to pH 8). This was followed by cleavage of the separated proteins by trypsin into peptide fragments and the analysis of the elution and washing solution by means of mass spectrometry (MS), whereby almost solely the peptides derived from bovine serum albumin were found in the elution solution.

[0110] Given that separation of proteins/peptides with cysteine/cysteines according to the invention is based on the specific interaction between —SH group or —SO.sub.3H group of cysteine and the oxide of metal/metals of the carrier according to the invention, it is evident that the other cysteine-containing peptides/proteins will behave in the same or similar manner if the same or similar technique of separation is used.

Separation of Nucleic Acids

[0111] Chains of deoxyribonucleic acids (DNA) are separated using a carrier according to the invention on the basis of the interaction between phosphate groups, which are part of nucleic acids, and the oxide of metal/metals of the carrier. The binding of DNA to the carrier occurs in a buffer based on the 2-(N-morpholino)ethanesulfonic acid (MES) in the pH range of 4.5 to 6.5. Afterwards, acetate buffer with a pH range of 3.8 to 4.5 is used for separation of ribonucleic acids (RNA). Generally, in both cases it is possible to use any buffer or any substance which, reaching the particular range of pH, is not at the same time an elution agent. Preferably, washing solutions for removing undesirable and non-specifically bound components are in both cases identical with the binding solutions in which the binding to the carrier takes place.

[0112] Elution is in both cases performed by increasing pH to a value of 8-11, or by an elution solution containing at least 20 mM of phosphate/phosphates, e.g. disodium phosphate or phosphate buffer.

[0113] The analysis of the elution and washing solutions is then carried out e.g. by means of electrophoresis (agarose or polyacrylamide) or by means of polymerase chain reaction (PCR).

[0114] Separation of nucleic acids is indicated in FIG. 2 by the first flow chart on the right-hand side.

Example 6

[0115] 1 mg of the carrier according to the invention, which was previously stored in a solution containing 80% of ACN and 0.1% of TFA, was under the influence of a magnetic field washed with 2×500 μl of a washing solution containing 100 mM of MES buffer with pH 5.5. To the washed carrier was then added a binding solution containing 10 μg of model deoxyribonucleic acid-oligonucleotide in 100 μl 100 mM of MES buffer with pH 5.0. The carrier with the applied binding solution was afterwards incubated under gentle rotation at room temperature for 60 min.

[0116] After incubation, the binding solution with unbound components was removed from the test tube and the carrier was washed with 5×200 μl of a washing solution containing 100 mM of MES buffer with pH 5.5.

[0117] 100 μl of an elution solution Na.sub.2HPO.sub.3 was then added to the carrier thus washed, elution proceeded under rotation for a period of 15 min. After its completion, both the elution and the washing solution were removed and analyzed by polyacrylamide gel electrophoresis using fluorescent detection, in which it was found that not only can oligonucleotides be bound to the carrier according to the invention, but they can also be easily released from it.

Regeneration of the Carrier

[0118] So as to regenerate the carrier according to the invention with the purpose of reusing it, it is necessary to remove all residues of organic substances from the carrier. In the case of the carrier which contains a TiO.sub.2 core it is possible to use for this purpose preferably irradiation of the carrier by UV radiation in an aqueous medium. TiO.sub.2 in that case generates on its surface highly reactive hydroxyl radicals, which have a considerable ability to cleave organic molecules into elemental carbon dioxide and water, by virtue of which the carrier according to the invention is completely free from any kind of undesirable organic residues. At the same time, however, there is no degradation of the layers or nanoparticles of magnetic metal oxide/oxides (e.g. Fe.sub.3O.sub.4), and so the magnetic properties of the carrier remain unaffected.

[0119] This form of regeneration is possible in the case of other types of a carrier according to the invention with cores formed by other transition metals.

[0120] In order to achieve the maximum effect of UV radiation, it is advantageous if the carrier is dispersed in an aqueous solution circulating in a closed flow system through a silicone tube, in front of which is situated a source of UV radiation with appropriate wavelength. The flow rate in the system must be selected with respect to the diameter of the silicone tube and the length of the flow system so that the carrier will be exposed to the radiation for at least 25% of the total time.

[0121] In another variant, it is also possible to use a batch system, e.g. a suitable vessel with a carrier in an aqueous solution. In that case, it is necessary to use mechanical stirring, e.g., propeller stirring or rotation on a rotator, and avoid using a magnetic stirrer which would cause clustering of the carrier at the point of the action of the magnetic forces.

[0122] After the completion of photocatalytic degradation it is then possible to separate the carrier from the aqueous medium by the magnetic field.

[0123] For photocatalytic degradation of residues of organic substances it is advantageous to use UV radiation having a wavelength of 200-400 nm, preferably in the range between 260 and 340 nm. In addition, the intensity of UV radiation must be at least 1 mW/cm.sup.2 of the irradiated area of the carrier, whereby the intensity 60 mW/cm.sup.2 of the irradiated area of the carrier enables complete decomposition of organic molecules within 80 minutes and the intensity 300 mW/cm.sup.2 of the irradiated area of the carrier within 12 minutes.