DISPERSION USING A MOVING MAGNET

Abstract

This invention relates to a method of dispersing magnetic particles, said method comprising or consisting of: (a) in a vessel, combining at least one permanent magnet and said magnetic particles in a liquid phase; and (b) triggering a fluctuating or oscillating motion of said permanent magnet using a magnetic field; thereby dispersing said particles.

Claims

1. A method of dispersing magnetic particles, said method comprising: (a) in a vessel, combining at least one permanent magnet and said magnetic particles in a liquid phase; and (b) triggering a fluctuating or oscillating motion of said permanent magnet using a magnetic field; thereby dispersing said particles.

2. The method of claim 1, wherein said magnetic particles carry at least one moiety on their surface, wherein said moiety is selected from: (i) a moiety capable of binding a target molecule, said moiety being a binding protein, an affinity chromatography material, an absorbing material, an adsorbing material, a probe, or a primer; (ii) a moiety capable of converting at least one starting molecule into at least one product molecule, said moiety being an enzyme or a chemical catalyst; and (iii) a moiety capable of forming an adduct with said target molecule; wherein said target molecule or said starting molecule, respectively, is present or suspected to be present in said liquid phase.

3. The method of claim 1, further comprising: (c) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (d) removing said liquid phase.

4. A method of separating a compound from a mixture, said method comprising: (a) in a vessel, bringing said mixture into contact with magnetic particles and at least one permanent magnet, wherein said magnetic particles carry a moiety capable of binding said compound; and (b) triggering a fluctuating or oscillating motion of said permanent magnet using a magnetic field; thereby separating said compound from said mixture.

5. The method of claim 4, further comprising: (c) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (d) removing remaining components of said mixture from said vessel.

6. The method of claim 4, further comprising: (e) adding a washing solution to said vessel; (f) triggering a fluctuating or oscillating motion of said permanent magnet using said magnetic field; (g) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (h) removing said washing solution from said vessel; wherein steps (e) to (h) may be repeated.

7. The method of claim 5, further comprising: (i) adding an eluent to said vessel, wherein said eluent reduces or abolishes binding of said compound to said moiety; (j) triggering a fluctuating or oscillating motion of said permanent magnet using said magnetic field; (k) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (l) separating the obtained eluate from said magnetic particles and said permanent magnet.

8. A method of producing at least one product molecule, said method comprising: (a) in a vessel, bringing at least one starting molecule into contact with magnetic particles and at least one permanent magnet, wherein said magnetic particles carry a moiety capable of converting said at least one starting molecule into at least one product molecule; and (b) triggering a fluctuating or oscillating motion of said permanent magnet using a magnetic field; thereby obtaining said at least one product molecule.

9. The method of claim 8, further comprising: (c) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (d) removing said at least one product molecule from said vessel.

10. A method of derivatizing a first compound, said method comprising: (a) in a vessel, bringing said first compound into contact with magnetic particles and at least one permanent magnet, wherein said magnetic particles carry a moiety capable of forming an adduct with said first compound; and (b) triggering a fluctuating or oscillating motion of said permanent magnet using a magnetic field; thereby obtaining said adduct.

11. The method of claim 10, further comprising: (c) removing, rendering static, and/or rendering said magnetic field incapable of triggering said motion; and allowing said magnetic particles to gather on said permanent magnet; and (d) removing material not bound to said particles from said vessel.

12. The method of claim 1, wherein said magnetic field is generated by at least one external magnet selected from: (1) an electromagnet, wherein a fluctuating or oscillating electric current flows through said electromagnet; or (2) a permanent magnet or an electromagnet, wherein said vessel and said further permanent magnet or said electromagnet are moved relative to each other in a fluctuating or oscillating manner.

13. (canceled)

14. A kit comprising: (iv) magnetic particles; (v) at least one permanent magnet; and (vi) at least one liquid reagent such as a buffer.

15. A device comprising: (vii) means for generating a magnetic field; and (viii) a control unit configured to modify said field over time.

16. The device of claim 15, wherein said means for generating a magnetic field is configured to receive a vessel such that said vessel is exposed to said magnetic field when in use.

17. The device of claim 16, wherein said device further comprises said vessel.

18. The device of claim 17, wherein said device further comprises, in said vessel, a permanent magnet and/or magnetic particles.

Description

[0193] The Figures show:

[0194] FIG. 1: “Novel System Handling” shows peptide identification by means of MS when handling magnetic particles during the preceding sample preparation in accordance with the present invention. In comparison, classic handling shows the number of distinct identified peptides when using an art-established magnetic separator.

[0195] The Examples illustrate the invention.

EXAMPLE 1

[0196] Performance Comparison with Prior Art Magnetic Separator

[0197] Material

[0198] Saccharomyces cerevisiae cell pellets with approximately 100 μg protein content were used for digestion tests. A permanent Neodymium magnet was used for beads handling (MagnetExpert; spheric, 2 mm). For lysis and clean-up, the iST-Kit including buffers and plastic ware was used (PreOmics GmbH, P.O.00001). Trypsin magnetic microparticles were provided by ReSyn Biosciences (ReSyn Biosciences (Pty) Ltd). A Helmholtz-coil setup was used to generate an external, oscillating magnetic field.

[0199] Methods

[0200] Classical Magnetic Particle Handling

[0201] S. cerevisiae cell lysis was prepared as described in Standard iST sample preparation (PreOmics GmbH, iST-Kit, P.O.00001). 750 μg immobilized trypsin beads (Trypsin magnetic microparticles) were equilibrated with 2×70% ethanol; lx 1% ammoniumhydroxide 3×50 mM Tris, pH 8. For each equilibration step, beads were incubated at 500 rpm for 3 min and supernatant was removed using a magnetic separator and waiting for 30 s for the magnetic microparticles to collect. Equilibrated beads were mixed with denatured yeast samples. Digestion was performed at 37° C. and 500 rpm for 1 h. Prior to peptide purification and LC-MS analysis (see standard iST sample preparation), magnetic trypsin beads were removed from samples using the magnetic separator and 100 μl of stop buffer were added. After elution, purified peptides were dried in the SpeedVac and resuspended in 2% acetonitrile, 0.1% trifluoroacetic acid. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled with a Thermo LTQ Orbitrap XL. Peptide loads of 5 μg were separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/MS data was searched against a yeast database using the MaxQuant software with default settings except using unspecific search.

[0202] Novel System Magnetic Particle Handling

[0203] S. cerevisiae cell lysis was prepared as described in Standard iST sample preparation (PreOmics GmbH, iST-Kit, P.O.00001). 750 μg immobilized trypsin beads (Trypsin magnetic microparticles) were equilibrated with 2×70% ethanol, lx 1% ammonium hydroxide and 3×50 mM Tris, pH 8 on the Helmholtz coil setup with a 2 mm round Neodymium magnet. For each equilibration step, samples were incubated on the Helmholtz system for 3 min applying a square wave function at 120 Hz and supernatant was removed after turning the field off and letting the magnetic particles collect for 10 s. Equilibrated beads were mixed with denatured yeast samples and digestion was performed on the Helmholtz system for 60 min applying a square wave function at 120 Hz. Prior to peptide purification and LC-MS analysis (see Standard iST sample preparation), magnets and magnetic trypsin beads were removed from samples and 100 μl of stop buffer were added. After elution, purified peptides were dried in the SpeedVac and resuspended in 2% acetonitrile, 0.1% trifluoroacetic acid. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled with a Thermo LTQ Orbitrap XL. Peptide loads of 5 μg were separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/MS data was searched against a yeast database using the MaxQuant software with default settings except using unspecific search.

[0204] Results

[0205] See FIG. 1 and caption thereof.

[0206] Discussion

[0207] In terms of the number of peptides identifiable by MS, the method of the invention performs at least as good as the classical handling of magnetic particles. Yet, and as discussed herein above, the method of the invention confers distinct advantages such as there being no requirement for a mechanical movement of the magnet outside the vessel and the avoidance of any cross-contamination.

EXAMPLE 2

[0208] Materials

[0209] For cell pellets containing approximately 100 μg of yeast proteins, commercially available Saccharomyces cerevisiae was resuspended in H.sub.2O and aliquots of 1 ml with OD 0.6 at 600 nm were prepared and centrifuged. Standard sample preparation for LC-MS analysis was performed with the iST kit from PreOmics GmbH (P.O.00001) including buffers, enzymes and plasticware. A permanent Neodymium magnet was used for beads handling (MagnetExpert; spheric, 2 mm). ZrO.sub.2 magnetic microparticles were provided by ReSyn Biosciences (ReSyn Biosciences (Pty) Ltd). A Helmholtz-coil setup was used to generate an external, oscillating magnetic field. Peptide clean-up was performed using cation exchange cartridges.

[0210] Method

[0211] Sample preparation was carried out according to the PreOmics standard protocol (PreOmics GmbH, P.O.00001) for yeast samples, followed by phosphopeptide enrichment on magnetic microparticles (ReSyn Biosciences), and purification of the sample using the reversed-phase cartridges. Purified peptides were dried to completeness under vacuum at room temperature.

[0212] For the enrichment of the phosphopeptides, first the magnetic beads were equilibrated. A spheric Neodymium magnet with a radius of 2 mm was added to 10 μl of bead suspension. The suspension was allowed to clear and the supernatant was taken off. 50 μl of wash buffer (70% ethanol) was added and the sample mixed by using an external magnetic oscillating field (120 Hz, square wave form, 5 min). After that, the suspension was allowed to clear and the supernatant was taken off. This procedure was repeated once for a total of two washes with this buffer.

[0213] 50 μl of 1% ammonium hydroxide was added and the sample was mixed again using the method of the invention (120 Hz, square wave form, 10 min). Again, the sample was allowed to clear, and the supernatant was taken off. The beads were then equilibrated three times using 50 μl of 0.2 M glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile using the same procedure as above.

[0214] Then 100 μl of 0.2 m glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile was added to the dried peptide pellet from above and resuspended properly. The solution was then added to the equilibrated beads and incubated while mixing with the method of the invention (120 Hz, square wave form, 20 min). The sample was allowed to clear and the supernatant was taken off. Unbound sample was removed by adding another 50 μl of 0.2 M glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile and mixing with the method of the invention (120 Hz, square wave form, 2 min). The sample was allowed to clear, supernatant was taken off.

[0215] 100 ul of washing buffer (1% trifluoroacetic acid in 80% acetonitrile) was added and the sample was mixed with the method of the invention (120 Hz, square wave form, 2 min) followed by allowing the sample to clear and taking off the supernatant. This step was repeated by another washing buffer (0.2% trifluoroacetic acid in 10% acetonitrile).

[0216] The phosphopeptides were eluted from the magnetic beads by adding 40 μl elution buffer (1% ammonium hydroxide) and incubated with mixing using the method of the invention (120 Hz, square wave form, 5 min). The sample was allowed to clear, the supernatant was taken off and transferred to a fresh tube. The elution was repeated twice for a total elution volume of 120 μl.

[0217] The tube containing the elution was centrifuged at maximum speed to pellet fragments of the magnetic beads. Using a magnetic separator, the supernatant was taken off and transferred to a new tube. The sample was dried under vacuum at room temperature until completely dry.

[0218] Next, a cation-exchange cartridge was equilibrated by adding 200 μl methanol followed by centrifugation in a waste tube (3.800 rcf, 1 min). A second equilibration was performed using 200 μl 2% acetonitrile, 0.1% trifluoroacetic acid buffer as described above.

[0219] The dried phosphopeptides were resuspended in 200 μl 2% acetonitrile, 0.1% trifluoroacetic acid buffer and loaded on the cartridge and centrifuged (3.800 rcf, 1 min). Then 200 μl 0.1% formic acid was added followed by centrifugation (3.800 rcf, 1 min) for washing the bound peptides. This washing step was repeated twice. The cartridge was transferred to a fresh collection tube, 200 μl of 0.1% formic acid in 80% acetonitrile was added followed by centrifugation (3.800 rcf, 1 min) for eluting the peptides from the cartridge. The elution step was repeated once for a total of two elution steps. The samples were dried under vacuum at room temperature until completely dry and then resuspended in 6 μl 2% acetonitrile, 0.1% trifluoroacetic acid buffer.

[0220] Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled with a Thermo LTQ Orbitrap XL. Peptides were separated on a home-made C18 column applying a 45 min gradient and tandem mass spectrometry was performed using a DDA Top 10 method. The MS/MS data was searched against a yeast database using the MaxQuant software with default settings.

[0221] Results

TABLE-US-00001 Proteins identified Peptides identified Phosphopeptides (STY) [%] 277 419 60.632

[0222] Discussion

[0223] The specificity apparent from the results demonstrates that the method of the invention satisfactorily performs for separation and enrichment purposes.

EXAMPLE 3: DISPERSION OF MAGNETIC PARTICLES USING DIFFERENT WAVEFORMS

[0224] Materials

[0225] A permanent Neodynium magnet with Palylene coating was used (cylindric; 2 mm×2 mm). For magnetic microparticles, 3.0 μm Amine-functionalized beads were used. A Helmholtz coil was used to generate an external, oscillating magnetic field. For creating different waveforms at defined frequencies, an Online Tone Generator (https://onlinetonegenerator.com/) was used.

[0226] Methods

[0227] Magnetic particles were resuspended by mixing thoroughly for 1 min. Three aliquots with each 50 μl of the particles were transferred into 0.5 ml screwcap tubes. The material was washed once with and resuspended in 50 μl ultrapure water (LC-MS grade; Fisher Scientific). One magnet per tube was added. The tubes were transferred to Helmholtz coils and repeatedly for 30 sec. each a square wave function, a sinoid function, a sawtooth or a triangle function was applied at 1-140 Hz. After turning the field off, the magnetic particles were allowed to settle on the magnet for 30 s.

[0228] Results

[0229] For all of the applied waveforms, it was found that the magnetic particles were mixed in the vessels while the magnetic field was turned on. The magnetic beads settled down on the magnets in the vessels when the magnetic field was turned off. Differences in mixing effect could be observed (see table 1).

TABLE-US-00002 TABLE 1 Mixing effect of different frequencies at different waveforms Waveform Frequency (Hz) Mixing effect Square 1 to 3 slight mixing 4 to 10 good 11 to 85 very good 90 to 140 good Sine 1 to 10 slight mixing 11 to 22 good 23 to 140 very good Sawtooth 1 to 4 good 5 to 119 good 120 to 140 good Triangle 1 to 9 slight mixing 10 to 24 good 25 to 140 very good

[0230] Discussion

[0231] The experiment demonstrates that dispersion of magnetic particles can be done with different waveforms at different frequencies.

EXAMPLE 4: DISPERSION OF MAGNETIC PARTICLES WITH DIFFERENT COILS

[0232] Materials

[0233] A permanent Neodynium magnet with Parylene coating was used (cylindric; 2 mm×2 mm). For magnetic microparticles, 3.0 μM Amine-functionalized beads were used A simple coil (as opposed to a Helmholtz coil) setup (8 small coils in a row; diameter of one coil 1.1 cm; height 2.0 cm) was used to generate an external, oscillating magnetic field. For creating different waveforms at defined frequencies, an Online Tone Generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W).

[0234] Methods

[0235] Magnetic particles were resuspended by mixing thoroughly for 1 min. Three aliquots with each 50 μL of the particles were transferred into 1.5 ml Eppendorf reaction vessels. The material was washed once with and resuspended in 50 μl ultrapure water (LC-MS grade; Fisher Scientific). One magnet per tube was added.

[0236] The reaction vessels were placed in the coil setup (one vessel per coil). The samples were incubated repeatedly for times of 30 sec. applying a square wave function, a sinoid function, a sawtooth or a triangle function at 1-180 Hz. After turning the field off, the magnetic particles were allowed to settle on the magnet for 10-30 sec.

[0237] Results

[0238] It was found that the magnetic particles were mixed in the vessels in a single coil setup while the magnetic field was turned on. The magnetic beads settled down on the magnets in the vessels when the magnetic field was turned off.

[0239] Differences in mixing effect at different frequencies could be observed (see table 2).

TABLE-US-00003 TABLE 2 Mixing effect of different frequencies at different waveforms Waveform Frequency (Hz) Mixing effect Square 1 to 4 slight mixing 5 to 169 very good 170 to 180 good Sine 1 to 13 slight mixing 14 to 16 good 17 to 160 very good 161 to 180 good Sawtooth 1 to 9 good 10 to 160 very good 161 to 180 good Triangle 1 to 4 slight mixing 5 to 17 good 18 to 170 very good 171 to 180 good

[0240] Discussion

[0241] The experiment showed that dispersion of magnetic particles can be done with a simple coil setup, i.e., without requiring a Helmholtz coil, at different frequency ranges and waveforms.