Dispersion using moving magnet

Abstract

To provide improved means and methods of separation or synthesis by magnetic means.SOLUTION: A method of dispersing magnetic particles is provided, comprising or consisting of (a) combining at least one permanent magnet and the magnetic particles in a liquid phase in a vessel, and (b) triggering a fluctuating or oscillating motion of the permanent magnet using a magnetic field so as to disperse the particles.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for dispersing magnetic particles, comprising or consisting of: (a) combining said magnetic particles with at least one permanent magnet in a liquid phase in a vessel; and (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field, thereby dispersing said particles.

[0002] In this specification, a number of documents are cited, including patent applications and manufacturer's manuals.The disclosures of these documents are not considered relevant to the patentability of this invention, but are incorporated herein by reference in their entirety.More specifically, all reference documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Magnetic separation techniques are well established and have applications in, for example, chemical, life science and diagnostic applications. These methods rely on selectively dispersing or magnetizing functionalized nanometer to micrometer sized paramagnetic or ferromagnetic particles in a suspension setting. Equilibration, binding, reaction, washing or elution conditions can be applied while the particles are dispersed. At the magnetization stage, the particles and the supernatant can be separated.

[0004] Advantages of these methods include no volume limitations, the option of implementation as a direct batch method, and the ability to be used even in the presence of impurities associated with non-purification, such as cellular debris. The simple addition and removal of solid material independent of the target material is particularly suitable for DNA enrichment, for example, where the target DNA molecules can be extremely large. Furthermore, little or no hands-on work is required, and no time-consuming centrifugation steps are required. The ease of preventing potential cross-contamination and the ease of automation make magnetic separation techniques ideal for low- to high-throughput applications.

[0005] Magnetic separation of particles, regardless of application, usually needs to be performed repeatedly. Traditionally, many manual methods and most automated methods use a strong permanent magnet that is temporarily brought into close contact with the exterior surface of a vessel (typically a polymeric reaction tube in the range of 5 μL to 50 mL) in which the magnetic particles are held. The particles become magnetized and collect near the point where the magnetic field is strongest. This causes the magnetized particles to move out of the dispersion into aggregates or conglomerates. When very small particles are used, a stronger magnet generally needs to be used and / or the particle aggregation takes longer to complete, but this allows for a substantially complete recovery of the particles. This aggregation step relies on the magnetic force that attracts the particles to the magnetic field. To return the particles to the dispersion, the magnet is moved away from the vessel (and thus from the particles) and the particles can be dispersed again by mixing, e.g., pipetting up and down or shaking.

[0006] Non-permanent magnets can also be used as magnetic separation devices. Notably, PerkinElmer's Chemagic platform (PerkinElmer Chemagen Technologie GmbH) uses a system of transiently magnetized rods, where electromagnets are used to handle larger volumes, which are directly submerged in the solution containing the magnetic particles (Chemagic Magnetic Separation Module I (<10 ml); Magtration® System 8 liters x 7 ml).

[0007] All of these systems share the common feature of using physical motion to introduce or move a magnetization source towards the magnetic particles and remove the magnetization source away from the magnetic particles. Although the Chemagic platform mentioned above uses a different approach for magnetic separation, it requires the introduction of rods into the sample, which can lead to cross-contamination. Furthermore, the need for separate mixing and dispersion steps with different associated technical means is a feature shared with the prior art methods using magnets entirely outside the reaction vessel, discussed further above. Summary of the Invention

[0008] Considering the shortcomings of the prior art, the technical problem underlying the present invention can be seen as providing improved means and methods of manipulating magnetic particles. In this context, and depending on the particular envisaged application, the technical problem can be seen as providing improved means and methods of separation or synthesis, more particularly separation or synthesis by magnetic means.

[0009] This problem is solved by the subject matter of the claims. [Brief description of the drawings]

[0010] [Figure 1] "New system run" shows peptide identification by MS when magnetic particles are manipulated during prior sample preparation according to the present invention. In comparison, the traditional run shows the number of individual peptides identified when using magnetic separation devices established in the art. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Thus, in a first aspect, the present invention provides a method for dispersing magnetic particles, comprising or consisting of: (a) combining said magnetic particles with at least one permanent magnet in a liquid phase in a container; and (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field, thereby dispersing said particles.

[0012] Generally, magnetic particles in a liquid phase will settle due to gravity. This is detrimental to their interaction with any constituent of said liquid phase. For that reason, it is necessary to keep the magnetic particles in suspension to achieve efficient surface contact with the entire liquid phase. This is called "dispersion" and is classically achieved using mechanical means, e.g. shaking, vortexing or pipetting up and down. According to the present invention, dispersion is achieved by an oscillating or vibrating movement of said permanent magnet.

[0013] Magnetic particles (often referred to as magnetic microparticles; magnetic nanoparticles; magnetic beads; magnetic microspheres; paramagnetic, ferrimagnetic or ferromagnetic beads) are particles that respond to a magnetic field. Generally, they are paramagnetic or ferromagnetic.

[0014] A large number of functionalized magnetic particles are known in the art. The functionalization may be localized to the surface of the particle or, for example, in the case of porous particles, may extend to the internal surface within the pores.

[0015] Functionalization allows the magnetic particles to interact with substances in their vicinity, with or without inducing chemical modification of said substances. In other words, for example, there may be a non-covalent interaction between the analyte of interest and a moiety on the magnetic particle, which is also referred to as "binding" in the following. On the other hand, the magnetic particles may be equipped with moieties capable of inducing a chemical reaction that leads to the formation of a new chemical compound, which is referred to herein as the formation of a "product" or "adduct".

[0016] Preferred functionalizations, i.e. preferred moieties attached to the magnetic particles, are the subject of preferred embodiments disclosed further below.

[0017] In terms of size, the magnetic particles preferably have a diameter of 1 nm to 1 mm, or 100 nm to 60 μm. Preferably, for a given selection of magnetic particles, the size distribution is narrow. Also, chemically inert particles are preferred. This may be achieved by coating. Furthermore, physical stability has been an issue up until now. Thus, particles that do not break down under normal use conditions are preferred. The magnetic material contained in the magnetic particles may also be embedded in a matrix, such as silica or a polymer. This is also a means of imparting chemical and / or physical stability.

[0018] Generally, large numbers of magnetic particles are used. The actual number depends on the selected application and its scale. Exemplary values ​​are 100-100,000 particles per container, e.g., 1,000-10,000.

[0019] Prior to use, the magnetic particles are preferably equilibrated. If magnetic particles provided by one of the manufacturers listed further below are used, equilibration may follow guidelines provided by the manufacturer. Generally speaking, equilibration involves suspending the particles in an aqueous solution, such as a buffer solution.

[0020] The combining may be simultaneous and may be in any order.

[0021] The liquid phase is not particularly limited. Implementations include a sample or mixture that contains one or more compounds or analytes of interest. Analytes may belong to multiple classes. For example, a proteome is a collection of molecules that share the characteristic of being proteinaceous in nature. The term "liquid phase" encompasses solutions and suspensions.

[0022] The sample may contain further substances in addition to the molecule of interest. Such further substances may be contaminants which it is desired to remove. On the other hand, the liquid phase may also be pure. The liquid phase may be a solution of the compound of interest, without further constituents in addition to the compound of interest and the solvent.

[0023] An important feature of the liquid phase is that it provides an environment in which interactions can occur between the magnetic particles, more specifically, moieties on the surface of the particles, and any analytes or compounds of interest.

[0024] Magnetic particles are widely used, with applications ranging from analyte purification to solid-phase synthesis. To optimize the yield of these processes, reaction mixtures containing the analyte or starting material with the particles are mixed, which is conventionally typically done by pipetting up and down, shaking, or vortexing.

[0025] The inventors have unexpectedly found that adding at least one permanent magnet to the reaction mixture and inducing movement of said permanent magnet by a magnetic field generated by a further magnet located outside the vessel holding the reaction mixture is an effective and convenient means of mixing and confers a number of distinct advantages.

[0026] It is notable that the prior art approaches also use magnets to manipulate the magnetic particles, i.e., to concentrate them in one or a few defined locations inside the vessel so that any remaining liquid containing unbound material can be removed. However, the prior art approaches do not use magnets for mixing. Rather, magnets are absent during the mixing phase.

[0027] Thus, the contribution this invention makes to the art is the use of an element, a magnet inside a vessel that holds a reaction mixture with magnetic particles, not only to collect the particles, but particularly to mix the reaction mixture.

[0028] The use of magnets for mixing also makes the mixing process more efficient compared to pipetting up and down or shaking. Specifically, the permanent magnets, by their own fluctuating or vibrating motion, make the flow (in the rheological sense) of the liquid reaction mixture more turbulent. Furthermore, the attractive forces between the magnetic particles and the permanent magnets cause the movement of the magnetic particles to be out of phase with the movement of the liquid. It is noteworthy that the magnetic force exerted by the external magnet is absent in the prior art mixing processes, which provides the above-mentioned advantages.

[0029] The examples contained herein evaluate the performance of the method using the number of peptides identified in subsequent analysis by mass spectrometry, and as demonstrated, the present invention works at least as well as conventional magnetic particle manipulations while providing the advantages discussed above.

[0030] The containers disclosed above are not particularly limited.

[0031] Useful containers include those commonly used in the fields of molecular biology and in vitro diagnostics. Such containers generally have surfaces that are free or substantially free of contaminants, chemically inert, and / or have low binding capacity.

[0032] It is important in the context of the present invention that the container has at least one wall that does not shield magnetic fields.Preferably, the entire container is made of a material that does not shield magnetic fields.Suitable materials include plastics, polymers, such as polypropylene, glass and ceramics.Metals can also be used, keeping in mind the requirement of magnetic permeability.

[0033] Exemplary and preferred containers are configured to hold volumes of 5 μL to 1 L, preferably 10 μL to 50 mL, and more preferably any of the following volumes: 30 μL, 40 μL, 100 μL, 150 μL, 200 μL, 250 μL, 500 μL, 1 mL, 1.5 mL, 2 mL, 5 mL, 15 mL, and 50 mL.

[0034] The vessels may be arranged in an array, for example in a common format (eg, a one- or two-dimensional array having 6, 24, 96, 384 or 1546 wells).

[0035] The vessel may also be implemented as a microfluidic device, ie a miniaturized device comprising one or more channels, optionally with openings having extensions or containers and / or valves.

[0036] The term "vessel" further encompasses vessels with a closed bottom, vessels with a lid, vessels with a closed bottom and lid, completely closed or sealed vessels, tubular elements, and elements with at least two openings allowing a continuous flow of the liquid phase through such elements, and in such flow-through vessels or reactors, said at least one permanent magnet and its movement are controlled by a magnetic field.

[0037] Preferably, the magnetic field is generated by a magnet that is external to the vessel. An external magnet can be implemented in various ways. See below for further details. Generally speaking, what is important is that the magnetic field is capable of inducing an oscillatory or vibratory movement of a permanent magnet located inside the vessel. This can be achieved by a time-varying magnetic field at the site of the vessel. Such a change over time can be achieved by movement of the elements that generate the magnetic field in space. Alternatively, or in addition, this can be done by changing a magnetic field generating parameter over time, for example by changing the current through a conductor, e.g. a coil, over time.

[0038] Thus, the magnetic field may be generated by an electromagnet, in which case the current through the electromagnet varies with time, such that under the influence of the magnetic field at least one permanent magnet undergoes an oscillating or vibrating motion, e.g. as required for mixing.

[0039] Said magnetic field may be generated by a permanent magnet, and if at least one permanent magnet is inside the vessel, in such a situation said permanent magnet is a further permanent magnet, and this "external" permanent magnet may undergo a movement in three-dimensional space, so that the magnetic field at the site of the vessel generated by said external permanent magnet changes over time, again with a view to generating said fluctuating or oscillating movement.

[0040] A permanent magnet is a piece of ferrimagnetic or ferromagnetic material.

[0041] The size of the single magnets may vary widely. As far as permanent magnets inside the vessel are concerned, these may be appropriately selected depending on the dimensions of the vessel, or equivalently, the reactor, to be used.

[0042] In terms of relative size, it is preferred that the largest dimension of the permanent magnet passes through the smallest passage or cross section in the reactor or vessel. Smaller than the smallest dimension preferably means 2 / 3, 1 / 2, 1 / 3, 1 / 4, or 10% of the smallest dimension or cross section of the vessel. Such mechanisms generally provide free or substantially free motion. Preferably, the free or substantially free motion occurs around or along at least two, at least three, at least four, at least five, or preferably all six axes of translation and rotation, and preferably the free or substantially free motion includes translation along at least two axes.

[0043] To describe a point-like object, there are three degrees of freedom of motion, i.e. translations in three independent directions spanning three-dimensional space. For extended objects, there are three additional degrees of freedom that can be defined with respect to three independent axes of rotation.

[0044] For completeness, exemplary values ​​of useful permanent magnet sizes are set forth herein from 0.1 mm to 10 cm, e.g., 0.2 mm to 2 cm (including any of the following values ​​and ranges defined thereby: 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, and 1 cm). The same preferred sizes and size ranges apply to any permanent magnets contemplated herein, and also to implementations in which multiple magnets are used. Generally, these lengths refer to the maximum extension of the permanent magnet.

[0045] Alternatively, multiple permanent magnets, such as those defined above, can be used. Exemplary but non-limiting numbers are in the single and double digit range, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10. More than 10 magnets can also be used, such as 20, 30, 40, or 50 magnets. These numbers refer to one container, even when a container arrangement (e.g., a microtiter plate) is used.

[0046] It will be understood that the number of permanent magnets is limited only as it is specified, in other words, while the method of the first aspect may include other strategies, such possibilities do not extend to the option of there being more permanent magnets than are expressly specified.

[0047] Such multiple magnets may be identical to one another or may be different.

[0048] The same generally applies to magnets, insofar as multiple magnets should be used. To take a specific example, a single magnet may be combined with a single ferromagnetic or ferrimagnetic bead. This provides for more precise movement of the magnet, while the parameters controlling the magnetic field remain unchanged. See also below.

[0049] A further means of achieving more precise movement is the addition of one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10, beads made of a material that cannot be magnetized, e.g., a non-magnetic metal. Particularly preferred is one such bead and / or a plurality of such beads of the same size or comparable size to the permanent magnet.

[0050] Regarding the shape of the permanent magnet, there is no particular limitation, and preference is given to shapes that do not negatively impede the free movement of the magnet. Exemplary shapes include sticks, bars, rods, blunt rods, cubes, rectangular prisms, prisms, spheres, oblate and flat ellipsoids, disks, tetrahedrons, octahedrons, dodecahedrons, and icosahedrons.

[0051] In a further preferred embodiment, (a) said permanent magnet comprises or consists of a ferromagnetic or ferrimagnetic material; and / or (b) said magnet and / or said particles are preferably coated with (i) a coating that confers chemical stability; (ii) a coating that confers mechanical stability or hardness; (iii) a coating with a catalyst; (iv) a coating with nucleic acids, e.g. probes and / or primers; (v) a coating with chelating agents, e.g. IMAC, TiO2 and ZrO2; (vi) preferably with (1) reversed-phase groups, e.g. C18, C8, benzene; (2) HILIC groups, e.g. hydroxyl groups; (3) cation exchange groups, e.g. sulfonic acid, phosphoric acid, carboxylic acid; (4) anion exchange groups, e.g. primary, secondary, tertiary and quaternary ions. (vii) preferably a globulin, particularly an immunoglobulin; an antigen, preferably an antigen capable of binding to an immunoglobulin; a ligand binding protein and / or their cognate ligand selected from streptavidin, biotin; protein A, protein G; annexin V, phospholipids; enzymes, such as oxidoreductases, transferases, ligases, such as polymerases, hydrolases, such as proteases, peptidases, nucleases, saccharidases, lipases, lyases and isomerases; a lipid coating; and (viii) coated with a coating selected from any one of a combination of (i) to (vii).

[0052] Suitable materials for the permanent magnet include the following elements and their alloys: neodymium-iron, neodymium-iron-boron (e.g., NdFe 14 B), cobalt, gadolinium, terbium, dysprosium, iron, nickel, iron oxide, manganese-bismuth, manganese-antimony, manganese-arsenic, yttrium-iron oxide, chromium oxide, europium oxide, and samarium-cobalt. Particularly preferred materials are neodymium-iron and samarium-cobalt.

[0053] Suitable coatings according to (c)(i) include polypropylene, polyethylene, polystyrene, parylene, titanium nitride, polyimides, chloropolymers, and fluoropolymers, preferably polytetrafluoroethylene (PTFE).

[0054] The at least one permanent magnet performs a fluctuating or oscillating motion, preferably the motion is induced by a fluctuating or oscillating magnetic field, preferably the magnetic field is induced by a current and and / or generated by electromagnets.

[0055] Simply put, the permanent magnet moves up and down and back and forth, the motion can have regular or repetitive components, but does not have to have them, and the spatial direction is not particularly limited. The permanent magnet may also rotate about one or more axes, in addition to the normal translational motion. The permanent magnet may hit or repeatedly hit the wall of the vessel, but does not have to do so. The motion of the permanent magnet is preferably not a directional motion. The motion is also, although possibly irregular, generally performed with respect to an average position located in the vessel, and the permanent magnet does not leave the vessel. The motion of the permanent magnet generally has one or more translational components, and the average position may be around the center of the vessel. Thus, unlike the motion performed by a magnetic stirrer, which is a rotation, the average position of the magnet is at or near the bottom of the vessel containing the liquid to be mixed or stirred.

[0056] The term "vibration" refers to regular motion, whereas "fluctuation" is broader and includes irregular motion as well. No particular preference is given in that respect.

[0057] The movement of the at least one permanent magnet is preferably induced by a varying or oscillating magnetic field.

[0058] Magnetic fields are a common means of controlling the position and / or movement of magnets. Given that permanent magnets are moved in accordance with the present invention, a fluctuating or oscillating magnetic field is used in this preferred embodiment.

[0059] Said magnetic field generated by an electric current is preferred. It is well established that electric and magnetic fields are interrelated, in particular that electric current generates a magnetic field. As a result, controlling the electric current is a means of controlling the magnetic field generated thereby.

[0060] Alternatively, or additionally, but less preferably, the magnetic field may be generated or adjusted, respectively, by an external permanent magnet. The term "external" means that such magnet is not located within the vessel. When an external magnet is used, the magnetic field generated thereby may be varied or oscillated by a corresponding movement of the external magnet with respect to the at least one permanent magnet inside the vessel.

[0061] In a preferred embodiment, the magnetic field is generated by an electromagnet. As used herein, the term "electromagnet" in its simplest implementation encompasses a portion of an electrical conductor through which an electric current flows in use. For better control of the magnetic field or for the purpose of generating stronger magnetic fields, specific implementations of electromagnets are envisaged, which are the subject of preferred embodiments disclosed further below.

[0062] In a preferred embodiment, the current fluctuates or oscillates. This behavior may also be referred to as a general "wave." The amount of current is known as amperage.

[0063] In preferred embodiments, the amperage of the current as a function of time is (i) a rectangular function, (ii) a sinusoidal function, (iii) a triangular function, (iv) a sawtooth function, or (v) a combination or convolution of any one of (i)-(iv). The time profile of the current is also referred to herein as a "waveform."

[0064] Considering that the current oscillates or fluctuates, this also applies to patterns (i)-(v), i.e., the rectangular and triangular functions are in fact repeating rectangular and triangular functions. The term "pattern" means that a given underlying event is repeated at least once. It refers to a sequence of events that is repeated. In a broader sense, the repetition need not be an exact repetition, for example the length of the rectangle in the time graph may vary (which is effectively a change in frequency; the preferred frequency as well as the preferred time dependence of the frequency are specified further below).

[0065] All of the embodiments (i)-(v) are also referred to herein as "alternating current."

[0066] Particularly preferred is the pattern of the rectangular function (also called rectangular wave or square wave), more particularly a repetitive rectangular function: surprisingly, the inventors have found that this pattern induces a particularly vigorous movement of the at least one permanent magnet, which is particularly efficient in terms of mixing.

[0067] However, other time profiles (waveforms) of current also work well, see Example 3.

[0068] In the above rectangular function, the time intervals of high and low current (or current off) may be the same or different. Means of controlling the length of the time intervals are known to those skilled in the art, such as what is called pulse width modulation (PWM). It is noteworthy that the energy transferred to the reaction mixture is not only governed by the frequency and amplitude of the current, but also by the relative duration of the time intervals.

[0069] The energy in a magnetic field per unit volume is E mag =1 / 2 B 2 / μ0, for the definition of B and μ0, see further below. magis equal to or less than the energy of the electric current that causes the magnetic field. The latter energy is E curr =UIt, where U is the voltage, I is the amperage of the current that creates the magnetic field, and t is the time that the current flows. In other words, controlling any one of B, U, I, and t is a means of controlling the amount of energy transferred by the permanent magnet to the contents of the vessel.

[0070] However, there are other means of specifying the details of the implementation of the method of the first embodiment. This includes specifying one or more of the parameters that can be more directly controlled or measured. These parameters include the frequency and amperage of the current, and may further include the dimensions of the vessel and coil, if used. Also, the strength of the magnetic field, preferably at the site of the permanent magnet, includes a means of quantitatively specifying the details of the implementation. Below, preferred ranges of the above-mentioned parameters are specified.

[0071] In a preferred embodiment, the current fluctuates or oscillates at a given frequency, preferably between 0.1 Hz and 20 MHz, more preferably between 10 Hz and 2 kHz, even more preferably between 50 and 500 Hz or between 90 and 300 Hz or between 100 and 200 Hz. It is understood that these scales apply not only to sinusoidal currents, but also to all current profiles specified herein, including, for example, repeating rectangular patterns. The term frequency can also apply to fluctuations, i.e. time-dependent behavior that is not regular (such regular time behavior is also referred to herein as "oscillation"), and is a means to characterize the time scale of the fluctuations. In such cases, the term "frequency" is understood to refer to the average frequency of the fluctuations.

[0072] For vessels having volumes in at least the single to double digit mL range, as well as for the wells of a standard 96-well plate, lower frequencies of 80 to 300 Hz work particularly well, whereas significantly higher frequencies, e.g., around 1000 Hz, may not induce vibration of the permanent magnet while at the same time inducing a full range of motion that encompasses a significant portion of the vessel volume. This does not mean that higher frequencies are not beneficial, and they may be used in conjunction with lower frequencies (see below).

[0073] More generally, the more preferred frequency ranges are those that ensure that the permanent magnets not only oscillate or rotate, but also perform a translational motion exploring the entire or substantially entire volume of the material to be processed in the method of the invention, said volume being generally the total volume of the liquid phase as contained in said vessel.

[0074] In other words, while guidance is given above for vessels with volumes in the single- to double-digit mL range and 96-well plates, the frequency range may require adaptation to vessels with significantly smaller volumes, significantly larger volumes, or special geometries. To give one example, for smaller volumes, such as wells of high-density microtiter plates (e.g., 1536-well plates), higher frequencies, such as about 1 kHz, e.g., greater than 200 Hz, are expected to cause permanent magnet motion comparable to that seen at smaller frequencies in larger vessels. In any case, those skilled in the art provided with the guidance given herein can explore and optimize the parameters that control the motion of the at least one permanent magnet in a straightforward manner. As further described above, permanent magnets that preferably perform translational motion in addition to rotation are preferred.

[0075] In a preferred embodiment, the frequency is kept constant throughout the performance of the method.

[0076] In an alternative preferred embodiment, the frequency varies as a function of time.

[0077] In a further preferred embodiment, more than one frequency is applied at a given time. In such a case, each frequency of such a plurality of frequencies may be selected from any of the preferred intervals given above. In the case of two frequencies, it is particularly preferred that the first frequency is between 50 Hz and 500 Hz and the second frequency is between 80 Hz and 20 MHz. In other words, this preferred embodiment provides a superposition of multiple frequencies.

[0078] More than one frequency includes 2, 3, 4, 5, 6, 7, 8, 9 and 10 different frequencies. Such multiple frequencies may be provided throughout instead of a single frequency. This means that such multiple frequencies are applied during the entire implementation of the method. Also, multiple frequencies, or different multiple frequencies, may be applied at different time intervals within a longer period. Within the longer period, there may also be one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 time intervals, in which only one frequency is applied, in addition to the time interval in which more than one frequency is applied.

[0079] In a further preferred embodiment, the frequencies are not constant over time, and in the case of more than one frequency, these frequencies are not constant over time but are preferably switched or gradually changed between two or more frequencies, preferably in a periodic manner.

[0080] An example of the control regime is (120Hz~1000Hz) n , (200Hz~1000Hz) n , or (100Hz~800Hz) n where n is an integer, for example from 2 to 1000, for example from 10 to 100, and specifies the number of times the frequency pattern in parentheses should be repeated.

[0081] The duration of the time interval when using constant frequency and / or constant amperage is: There is no particular limitation to the time interval, and a time interval of 1 second to 1 day, for example, 1 minute to 1 hour, is envisioned.

[0082] In a further preferred embodiment, the current (a) has an amperage I of 20 mA to 100 A, preferably 0.1 to 20 A, and (b) the magnet is rotated at a speed of 0.02 to 10 9 A / m, preferably 10 to 10 6 and / or (c) is exposed to a magnetic field strength of 100 nm to 100 nm A / m, and / or (b) is applied for a period t between 1 second and 1 week, for example between 10 minutes and 5 hours.

[0083] The magnetic field strength H determines the strength of the magnetic field and is measured in A per meter. H must be distinguished from the magnetic flux density B, which is particularly relevant in situations where a core is used to intensify the magnetic field of an electric current.

[0084] It should be noted that, as disclosed above, the amperage as a function of time varies on a time scale governed by the frequency disclosed herein, and there is no constant amperage with respect to the time scale of the variation. Furthermore, for practical purposes and consistent with the practice in electrodynamics, an alternating current can be quantified in terms of its average amperage. The above value is the average amperage in that sense. It is to be noted that the average is preferably greater than the time scale of the variation. That is, to the extent that an intermittent current is used, the average amperage is preferably within the ranges specified above when the current is on, and zero amperage is present when the current is off.

[0085] In preferred embodiments, the amplitude of the fluctuations or oscillations is (a) constant or (b) varies over time, preferably on a time scale slower than the time scale of said fluctuations or oscillations.

[0086] This embodiment refers to the amplitude of the current movement. The amplitude of the current oscillation or fluctuation is governed by amperage.

[0087] In a further preferred embodiment, the current is intermittent and / or the amperage is preferably varied over time in a periodic manner.This variation over time generally concerns a time scale slower than the time scale defined by the frequency of the alternating current.In other words, when an alternating current varies over time in the sense of this embodiment, the time dependence of the current is a superposition of two patterns or waves: the generally fast fluctuations inherent in alternating current, and generally slower variations.

[0088] An exemplary intermittent pattern is a repeating sequence of on (1 min)-off (1 min). Other preferred time intervals are given above. The advantage of the intermittent pattern is that it allows the temperature to remain constant or substantially constant, especially when the contents of the container are observed to heat up.

[0089] In a more preferred embodiment, a power of 0 to 1000 W, preferably 1 to 200 W, is applied.

[0090] In a further preferred embodiment, the current is supplied by a power supply, preferably having a potential or voltage U in the range of about 0 to 240 V, for example 0.1 to 75 V. These values ​​refer to the average voltage applied.

[0091] In a further preferred embodiment, the electromagnet comprises at least one coil, preferably comprising: (a) a plurality of, e.g., 1 to 10 4 and / or (b) having at least one Helmholtz coil, and / or (c) having at least one core.

[0092] Exemplary numbers of the plurality of coils are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 300, 400 and 500.

[0093] The term "Helmholtz coil" is established in the art and refers to an arrangement of usually two identical coils spaced apart such that their axes of rotational symmetry are aligned or occupy the same space. The magnetic field in the space between the coils is particularly homogeneous and / or particularly strong. Multiple Helmholtz coils may also be used, for example two Helmholtz coils.

[0094] However, the use of Helmholtz coils is not a requirement for the present invention to work successfully, see Example 4.

[0095] Further arrangements of multiple coils are known to provide extended spatial regions of particularly homogeneous magnetic fields. A further example is the Maxwell coil.

[0096] The core serves to intensify the effect of the magnetic field. The core is preferably made of a ferromagnetic material, such as iron, especially soft iron. The magnetic flux density B is related to the magnetic field strength as follows: B=μ r μH. μ is the magnetic permeability of a vacuum and is therefore a fundamental physical constant. On the other hand, μ r is the relative magnetic permeability, which determines the degree of enhancement of the magnetic flux density by a given material, e.g., a ferromagnetic core, under the influence of a magnetic field. Usually, μ for the ferromagnetic material to be used as the core is r is 10 3 ~10 6 , for example 200000 to 400000, for example about 300000. Suitable core materials include powdered metals, laminated metals, annealed metals, for example annealed iron, ceramics, and solid metals.

[0097] The preferred value of B in the absence of a core is 10 -8 ~10 4 Tesla (T), e.g. 10 -5 ∼1 T. If a core with a particular relative permeability is used, the value of B can be multiplied with the relative permeability. Thus, the preferred value of B in the presence of a core is 10 -2 ~10 9 , e.g., 1 to 10 6T. Generally, these values ​​refer to B at the site of the permanent magnet or within the vessel.

[0098] In terms of geometry, the preferred coil is circular. The preferred diameter is 1 mm to 1 m or 1 mm to 0.5 m, e.g. 2 mm to 300 mm or 2 mm to 200 mm. Also, coils of different geometries are envisaged, e.g. square, rectangular or triangular (i.e. all or part of the windings are square, rectangular or triangular). Finally, it is to be noted that a coil is not an essential requirement for an electromagnet, and also an arrangement of two anti-parallel wires may be used ("anti-parallel" referring to the direction of the current flowing through the two wires at a given time).

[0099] In a further preferred embodiment, more than one coil, preferably between 2 and 10 4 For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 coils, or 10 to 1000 coils may be used. It is understood that each coil may have one or more windings, with preferred numbers of windings being disclosed herein above.

[0100] In a preferred embodiment, the magnetic particle has at least one moiety on its surface, which is: (i) a moiety capable of binding to a target molecule, preferably a binding protein, an affinity chromatography material, an absorbent material, an adsorbent material, a probe, or a primer; (ii) a moiety capable of converting at least one starting molecule into at least one product molecule, preferably an enzyme or a chemical catalyst. and (iii) a moiety capable of forming an adduct with a target molecule, said target molecule or initiating molecule, respectively, being present or suspected to be present in said liquid phase.

[0101] Magnetic particles, both functionalized and non-functionalized, are available from a number of manufacturers, such as Resyn Biosciences (Pty) Ltd., Thermo Fisher Scientific, Creative Diagnostics, Nanopartz Inc., Alpha Nanotech Inc., Spherotech Inc., and Stratech. Those skilled in the art can also prepare functionalized magnetic particles using known procedures, see, for example, Sun et al., Current pharmaceutical biotechnology, vol. 10, pp. 753-60 (2009) and Perez-Ruiz et al., New Biotechnology, vol. 33, pp. 755-762 (2016).

[0102] The preparation of such magnetic particles is described, for example, in Shan et al., Langmuir, Vol. 22, pp. 2516-2522 (2006), Oberacker et al., Bio-protocol Vol. 9(No. 20): e3394, and in Hafeli U., Schutt W., Teller J., Zborowski M. (eds.) Scientific and Clinical Applications of Magnetic Carriers. Springer, Boston, MA, in Paulke BR, Buske N, Winoto-Morbach S. (1997) Synthesis Studies on Paramagnetic Polystyrene Latex Particles.

[0103] The target molecule or the initiating molecule is generally dissolved or suspended in the liquid phase.

[0104] An exemplary binding moiety according to item (i) of the above preferred embodiment is ZrO2. ZrO2 binds to phosphopeptides. Example 2 illustrates the use of magnetic particles having ZrO2 moieties according to the present invention.

[0105] Exemplary enzymes include trypsin and other proteases, such as LysC, GluC, AspN, ArgC or chymotrypsin, which cleave proteins, polypeptides and larger peptides into fragments that, due to their size, are suitable for downstream analytical methods, such as mass spectrometry (MS). Example 1 included herein illustrates the use of trypsin-coated magnetic particles in the context of both the practice of the invention and art-established methods of manipulating magnetic particles.

[0106] In a further preferred embodiment, the method of the first aspect further comprises or consists of: (c) removing, staticating and / or disabling the magnetic field from inducing the movement, causing the magnetic particles to collect on the permanent magnet; and (d) removing the liquid phase.

[0107] The phrase "further consisting of" in this specification refers to an implementation in which the method consists of a closed number of steps. In the case of the above embodiment, these are steps (a) and (b) as defined according to the first aspect and steps (c) and (d) which are the subject of the above embodiment.

[0108] Removal of the magnetic field may be accomplished by physically moving the external magnet away from the vessel such that the magnetic field generated by the external magnet becomes negligible at any location within the vessel. Alternatively, or in addition, this applies to implementations where the external magnet is an electromagnet, and the current through the electromagnet may be significantly reduced or turned off.

[0109] In a further alternative, the magnetic field is not removed or is not removed to a sufficient extent to stop the mixing / dispersion process. In that case, the magnetic field is changed so that it no longer induces the fluctuating or oscillating motion of the permanent magnet. As will be further disclosed below, generally, to induce such type of motion, the magnetic field is also fluctuating or oscillating in nature. By changing such an oscillating or fluctuating magnetic field to a static one, the motion of the permanent magnet stops. This is sufficient to stop the mixing process. Generally speaking, any modification of the magnetic field that makes it impossible to induce the motion is suitable to stop the mixing process.

[0110] Step (d) requires the removal of a liquid phase. It is noteworthy that the liquid phase at this stage may not be exactly the same as the liquid phase that was in the vessel prior to the addition of the magnetic particles. For example, if the liquid phase originally contained a compound or analyte that binds to moieties on the surface of the particles, the compound or analyte will be present in lesser amounts in the liquid phase to be removed in step (d) or will be absent from the liquid phase to be removed in step (d).

[0111] Generally, the assembly phase takes a few seconds, for example, 0.1 to 600 seconds, 1 to 60 seconds, or 10 to 30 seconds.

[0112] According to this preferred embodiment, the at least one permanent magnet is not only a means for dispersing the magnetic particles, but also serves to collect the particles when the magnetic vibration or fluctuation is absent or turned off. Many prior art approaches also use magnets to collect magnetic particles, but the magnets are generally external to the container and require additional devices to move the magnets closer to or away from the container. Another drawback of such prior art methods is that any container shields the magnetic field to at least some extent, and therefore theoretically possible magnetic flux density is not actually achieved inside the container. Thus, most prior art techniques require specially designed magnets with very high magnetic field strength.

[0113] The present invention differs from those approaches in that the at least one permanent magnet is located inside a vessel that holds a sample, a reaction mixture or a mixture to be purified.

[0114] The Chemagic method outlined above submerges a non-permanently magnetizable rod in the mixture, which exerts a magnetic field in the mixture by utilizing electromagnetism. However, the present invention is also different from this. That is, the at least one permanent magnet used by the present invention is submerged in the reaction mixture held in a vessel, rather than being integrated in a device that mechanically moves the magnetic rod. This difference makes the permanent magnet used in the present invention a disposable element. The risk of cross-contamination introduced by the magnetic rods mentioned above is avoided.

[0115] Although the method of the first embodiment is disclosed above by referring to at least one permanent magnet, it is noteworthy that any magnetic body may be used for that purpose. The term "magnetic body" as used herein refers to an assembly of particles, at least one of which is a magnet, and the assembly of said particles is mediated by the magnetic field of said at least one magnet. In short, in a magnetic field, the resulting assembly essentially behaves like a single magnet. For details, see further below. In terms of size, said particles are larger than the molecules to be fragmented (and smaller than the container or reactor in which said method is carried out).

[0116] In a second aspect, the present invention provides a method of separating a compound from a mixture, comprising: (a) contacting the mixture in a vessel with magnetic particles having a moiety capable of binding the compound and at least one permanent magnet; (b) separating the compound from the mixture using a magnetic field; and inducing a rocking or vibratory motion of the stone, thereby separating said compound from the rest of the constituents of said mixture.

[0117] As stated above, the oscillating or vibrating motion of the permanent magnet is a means of mixing, bringing the binding moieties into contact with the compounds, causing binding, and this provides the basis for separation of the liquid phase from the remainder of the composition.

[0118] This embodiment utilizes the advantages of the method of the first embodiment, but makes use of a particular type of functionalized particles, namely magnetic particles with binding moieties. As mentioned above, "binding" as used herein refers to a non-covalent interaction between a moiety on the particle and the compound of interest. Such particles are generally designed for separation processes, which can be analytical or preparative. As a result, the liquid phase of the first embodiment is implemented here as a mixture. That is, such a mixture may contain, in addition to the compound of interest (also referred to in some instances as "analyte"), other constituents (e.g. contaminants) that are not of interest or are undesirable, and the method of the second embodiment makes it possible to remove said other constituents, e.g. contaminants, and obtain the compound of interest in enriched or pure form. Generally speaking, the method of the second embodiment encompasses enrichment, depletion, purification, isolation, separation, fractionation, and clean-up procedures.

[0119] Preferred embodiments of the binding moiety include those disclosed above as preferred embodiments of the method of the first aspect. These preferred embodiments of the method of the first aspect define preferred embodiments of all aspects of the invention. Further preferred or exemplary moieties are disclosed further below and in the Examples contained herein.

[0120] An important difference from many prior art methods is that the magnets that control the movement and position of the magnetic particles are present in the reaction mixture rather than external to the vessel, providing the dual function of manipulating the magnetic particles and thoroughly mixing the reaction mixture from a single component.

[0121] Also, in stark contrast to established methods in the art, the magnet can be made into a totally disposable element, which facilitates avoiding carryover contamination between samples by the magnet.

[0122] Known devices for magnetic separation require a mechanical element to move the magnet, but this is not necessary by using an electromagnet as the external magnet, specifically, controlling the current through the electromagnet to induce the desired movement of a permanent magnet within the vessel.

[0123] In a preferred embodiment, the method further comprises or consists of: (c) removing, staticating, and / or disabling the magnetic field from inducing the movement, causing the magnetic particles to collect on the permanent magnet; and (d) removing the remainder of the components of the mixture from the container.

[0124] As noted above, this applies to all embodiments relating to removal of the magnetic field, which may be achieved by reducing or turning off the physical movement between the external magnet and the vessel and / or the fluctuating or oscillating current through the electromagnet. Furthermore, rather than being removed, the magnetic field may be rendered unable to induce movement of a permanent magnet inside the vessel, for example by making the magnetic field static.

[0125] Removal of the remainder of the composition, as well as removal of any other liquid from the container (e.g. removal of spent washing solution as disclosed below, or removal of eluates, also disclosed below) can be by any means and methods established in the art, such as pipetting.

[0126] The assembly of the particles onto the magnet generally occurs on a time scale measured in seconds. The corresponding preferred embodiments of the first aspect apply mutatis mutandis. However, the assembly time may depend to some extent on the selected settings, e.g. the magnetic field strength of the permanent magnet inside the container and the size of the magnetic particles. As will become clear further below, highly magnetic materials are preferred. Also, the permanent magnet is preferably uncoated, or if coated, the coating does not exert a strong shielding effect on the magnetic field generated by the permanent magnet.

[0127] In a preferred embodiment, the method further comprises or consists of: (e) adding a washing solution to the vessel; (f) inducing an oscillating or vibrating motion of the permanent magnet using the magnetic field; (g) removing the magnetic field, rendering it static and / or disabling the magnetic field from inducing the motion, causing the magnetic particles to collect on the permanent magnet; and (h) removing the washing solution from the vessel, and steps (e)-(h) may be repeated.

[0128] This embodiment relates to a washing procedure adapted to the manipulation of magnetic particles according to the invention. It therefore makes use of a permanent magnet inside the vessel and an external magnet. The same applies mutatis mutandis to the following preferred embodiment defining an optional elution procedure that finally delivers the compound of interest in the eluate.

[0129] To the extent that repeated washing is performed, this can be done with the same or different washing solutions, and an appropriate choice can be made by one skilled in the art depending on the particular application selected.

[0130] In a preferred embodiment, the method further comprises or consists of: (i) adding to the vessel an eluent that reduces or destroys binding of the compound to the moiety; (j) inducing an oscillating or vibrating motion of the permanent magnet using the magnetic field; and (k) removing the magnetic field, rendering it static, and / or disabling the magnetic field from inducing the motion, causing the magnetic particles to collect on the permanent magnet.

[0131] In a preferred embodiment, the method further comprises or further consists of (l) separating the resulting eluate from said magnetic particles and said permanent magnet.

[0132] In other words, an exemplary enrichment or clean-up process introduces magnetic particles into a sample containing the molecules or substances of interest; introduces a binding reaction by contacting the magnetic beads; introduces magnetic separation and collection of said magnetic particles on the magnet by adding a permanent magnet to the sample / magnetic particle dispersion; then conveniently removes the remaining liquid that no longer contains the target molecules and adds a washing solution; subsequently, introduces a fluctuating or oscillating magnetic field to disperse and effectively wash the magnetic particles; then, turns off the magnetic field to re-collect the particles and remove the wasted washing solution; finally, adds an elution buffer to the magnetic particles and reintroduces the fluctuating or oscillating magnetic field to perform an efficient elution; after turning off the fluctuating or oscillating magnetic field again, the eluate containing the purified molecules of interest can be further processed or analyzed.

[0133] In a preferred embodiment, the method further comprises or consists of one or both of the steps of: (a0) fragmenting the compound; and (a00) lysing cells containing the compound, if present, wherein step (a00), if performed, is achieved prior to steps (a0) and (a); and step (a0), if performed, is achieved prior to step (a).

[0134] This preferred embodiment is usually relevant when the starting mixture or liquid phase is more complex. "Complex" may refer to the matrix that embeds the analyte under consideration, which may be, for example, a large macromolecule of biological origin, and / or the matrix that embeds the analyte, which may be, for example, located within or on the surface of a biological cell or tissue.

[0135] Depending on the nature of the sample, step (a0) or both steps (a00) and (a0) may be of interest.

[0136] In a particularly preferred embodiment, step (a00) and / or step (a0) are achieved by inducing an oscillating or vibrating movement of said permanent magnet by means of said external magnet.

[0137] Depending on any downstream analysis that may be of interest, it may be desirable to fragment large macromolecules to produce smaller compounds. This is the case for most mass spectrometry sample measurements. As described in the applicant's previous patent applications EP20174469.5 and EP20174484.4, moving magnets are a useful means of fragmenting biopolymers, such as proteins or nucleic acids. This is the subject of step (a0) disclosed above. The same applies mutatis mutandis to instances in which the macromolecule is not the molecule of interest, but its inactivation is desired.

[0138] Similarly, as described in the applicant's previous patent applications WO2020 / 002577 and EP20174484.4, the moving magnet is also a means for disrupting biological cells and thereby making their contents available, which is the subject of step (a00) disclosed above.

[0139] In a third aspect, the present invention provides a method for producing at least one product molecule, comprising or consisting of: (a) contacting, in a vessel, at least one starting molecule with magnetic particles and at least one permanent magnet having a moiety capable of converting said at least one starting molecule into at least one product molecule; and (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field, thereby obtaining said at least one product molecule.

[0140] The method of the third embodiment utilizes functionalized magnetic particles with a catalyst on the surface. As further described above, such catalysts can be enzymes or chemical catalysts. The reaction catalyzed is not particularly limited. Examples include cleavage, e.g., hydrolysis, and synthesis reactions, e.g., polymerization.

[0141] In a preferred embodiment, the at least one product molecule does not remain bound to the particle, in other words, once the catalyzed reaction is complete, the product is free to diffuse away from the magnetic particle.

[0142] In a further preferred embodiment, the method further comprises or consists of: (c) removing the magnetic field, rendering it static, and / or rendering the magnetic field unable to induce the movement, causing the magnetic particles to collect on the permanent magnet; and (d) removing the at least one product molecule from the vessel.

[0143] In a fourth aspect, the present invention provides a method of derivatizing a first compound, comprising or consisting of: (a) contacting said first compound in a container with magnetic particles having a moiety capable of forming an adduct with said first compound and at least one permanent magnet; and (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field, thereby obtaining said adduct.

[0144] The method of the fourth embodiment utilizes magnetic particles having chemically reactive groups on their surfaces, where a chemical reaction occurring between the first compound and the reactive groups results in the formation of a covalent bond connecting the two.

[0145] The reactive group is not particularly limited, and can be selected by those skilled in the art without further ado according to the intended use.For example, when the first compound contains a primary amino group, the appropriate moiety is NHS ester.

[0146] The described adducts may also be referred to as conjugates. The adducts comprise or consist of the above compounds and the above moieties, both of which are connected by at least one covalent bond.

[0147] Preferably, the adduct remains attached to the particle.

[0148] It will be appreciated that the formation of such adducts is also a means of producing functionalized magnetic particles, in other words the method of the fourth aspect may be used as a method of producing functionalized magnetic particles.

[0149] Furthermore, the functionalized magnetic particles obtained via that route may be used as magnetic particles in the context of any of the aspects of the present invention.

[0150] Preferably, the method further comprises or consists of: (c) removing the magnetic field, rendering it static and / or rendering the magnetic field unable to induce the movement, causing the magnetic particles to collect on the permanent magnet; and (d) removing material that is not bound to the particles from the container.

[0151] In a preferred embodiment, the method further comprises or consists of: (e) adding a washing solution to the vessel; (f) inducing an oscillating or vibrating motion of the permanent magnet using the magnetic field; (g) removing the magnetic field, rendering it static and / or disabling the magnetic field from inducing the motion, causing the magnetic particles to collect on the permanent magnet; and (h) removing the washing solution from the vessel, and steps (e)-(h) may be repeated.

[0152] As is common in the art, washing steps in an analytical or preparative procedure may, but need not, be repeated, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. This applies to all aspects of the invention.

[0153] Related to the fourth aspect, the present invention provides in a fifth aspect a method of solid phase synthesis comprising carrying out once or repeatedly the method of the fourth aspect or any of its preferred embodiments, wherein said magnetic particles constitute the solid phase.

[0154] The number of iterations is not limited, it depends on the selected application, the desired properties of the product (e.g., length), and, as is common in synthetic procedures, the yield of each iteration and the desired yield of the final product.

[0155] In a preferred embodiment of the method of the fifth aspect, during the second and further performances of the method of the fourth aspect, second and further compounds are added to the vessel, respectively, thereby obtaining a product comprising the first, second and further compounds as components.

[0156] This preferred embodiment can be implemented as a progressive chain extension reaction, in which in a first round, an adduct is formed between the reactive moiety on the magnetic particle and a first compound, and in a second round, In the second round, a conjugate is formed between the adduct and a second compound.

[0157] This can be achieved, for example, by using a first compound which, in addition to a group reactive with a moiety on the magnetic particle, has a further group capable of forming a covalent bond with said second compound, either in situ or upon appropriate activation. This applies mutatis mutandis to further rounds insofar as they are carried out in the functioning of the method of the fifth aspect.

[0158] The resulting product may, but need not, have a continuous structure in the general form of first compound-second compound-third compound- etc. Depending on the compounds and / or the moieties on the magnetic particles, branched or crosslinked products may be obtained.

[0159] To avoid undesired side reactions, suitable protecting groups may be used. Removal of the protecting group occurs when the reaction of the protecting group occurs. Repeated cycles of protection and deprotection are established in the art, for example for the purpose of solid phase peptide synthesis, utilizing protecting groups such as fMoc and Boc.

[0160] In a preferred embodiment, the adduct or compound, respectively, is cleaved from the magnetic particle, for example by acid-, base- or light-induced cleavage of a covalent bond.

[0161] This is a procedure commonly used in solid phase synthesis, specifically, when the desired product is obtained, it remains bound to the solid phase, i.e., the magnetic particles, and is cleaved from the solid phase for further manipulation or processing.

[0162] The use of the present invention for synthesis purposes is not limited to the above-mentioned method of solid-phase synthesis, in which the first component is covalently bound to the particle (at least until the time when the final product is cleaved off).In fact, repeated rounds of synthesis may be carried out by using non-covalent interactions.An example of this is the hybridization between at least two nucleic acid species that occurs, for example, during the polymerase chain reaction (PCR).

[0163] It is understood that in the course of any such method involving multiple subsequent reactions, additional steps in addition to the steps explicitly described above may be beneficial or necessary. Such additional steps may be applied by those skilled in the art without further ado, depending on the particular synthesis or other method to be carried out. Examples of such additional steps include adding additional agents in addition to the compounds that ultimately form the building blocks of the synthetic molecule. Such additional agents include activating agents, blocking agents, neutralizing or pH shifting agents, and the like. Exemplary blocking agents are fMoc and Boc, as described above.

[0164] In a preferred embodiment of the method of any one of the above disclosed aspects, at least one ferromagnetic particle or bead is added together with the at least one permanent magnet. This is a preferred embodiment of the method of all aspects of the present invention. The inventors have surprisingly found that adding ferromagnetic particles or beads, preferably of a size comparable or identical to that of the permanent magnet, improves mixing by making the permanent magnet and the beads move more vigorously. In such a setup, multiple permanent magnets and / or multiple ferromagnetic beads may be used, but still, exactly one permanent magnet and exactly one ferromagnetic bead are preferred.

[0165] In preferred embodiments of all aspects of the method, one, more or all of steps (d), (e), (h), (i) and (l) are accomplished by pipetting.

[0166] Generally speaking, especially in such pipetting situations, it is advantageous to use still further magnets. In addition, during the phase where the magnetic field does not induce the oscillation or vibration of the permanent magnet inside the vessel and the magnetic particles are collected on the surface of the permanent magnet, they generally end up at the bottom of the vessel due to gravity. This can make pipetting less effective or more troublesome. Therefore, the further magnet, for example another permanent magnet, can be brought closer to the outer wall of the vessel. This allows the permanent magnet and magnetic particles inside the vessel to be located at the wall (inside the vessel). Now, when pipetting, the tip of the pipette can be inserted into the vessel and lowered right to the bottom.

[0167] In preferred embodiments of the methods of all aspects, the magnetic field is generated by an external magnet, preferably (1) an electromagnet, through which a fluctuating or oscillating current flows; and / or (2) a further permanent magnet or electromagnet, through which the vessel and the further permanent magnet or electromagnet are moved relative to one another in a fluctuating or oscillating manner.

[0168] Option (1) is particularly preferred.

[0169] In a preferred embodiment of the method of any one of the above disclosed aspects, the magnetic particles are paramagnetic, ferrimagnetic or ferromagnetic. A preferred material is magnetite. The magnetic particles do not have to be permanently magnetic. However, materials that are suitable materials for at least one permanent magnet are also suitable magnetic materials for the magnetic particles.

[0170] 10. The method of any one of the preceding claims, wherein the analyte or compound is a nucleic acid, ribonucleic acid, sugar, polysaccharide, protein, polypeptide, peptide, and / or lipid.

[0171] In a preferred embodiment of the method of any one of the preceding aspects, different steps are accomplished at different temperatures. Such a regime is useful for polymerase chain reaction (PCR), enzymatic processes using enzymes that have optimum temperatures above ambient temperature, or where elevated temperatures interfere with, e.g., are a means to reduce, binding.

[0172] 13. The method of any one of the preceding claims, wherein a plurality of vessels, such as a microtiter plate of vessels, are processed simultaneously.

[0173] In connection with all previous aspects, the present invention further relates to the use of at least one permanent magnet, magnetic particles and a magnetic field for mixing a reaction mixture in a vessel, wherein said at least one permanent magnet and said particles are within said vessel, preferably said magnetic field being generated by a further magnet external to said vessel.

[0174] As described in connection with the method of the present invention, said further magnet may be implemented as a permanent magnet or an electromagnet, for example a coil.

[0175] In a further aspect, the present invention provides elements suitable for carrying out the methods and uses of the present invention as a kit, said kit comprising or consisting of: (i) magnetic particles; (ii) at least one permanent magnet; and (iii) at least one liquid reagent, e.g. a buffer solution.

[0176] In a preferred embodiment, the magnetic particles are preferably functionalized with binding and / or reactive moieties.

[0177] Generally speaking, the preferred embodiments of the method of the invention also define the preferred embodiments of the use, kits and devices of the invention. For example, in preferred embodiments, the kits In addition to the at least one permanent magnet as described above, the sample comprises at least one ferromagnetic or non-magnetic bead, as described in more detail in connection with the methods of the present invention.

[0178] In a further preferred embodiment, the kit further comprises or further consists of (iv) a manual containing instructions for carrying out the method of any one of the first to fifth aspects.

[0179] In a further preferred embodiment, the kit further comprises or further consists of at least one container or at least one array of containers, said container or array of containers being preferably as defined herein above.

[0180] In a further aspect, the present invention provides a device comprising or consisting of: (i) means for generating a magnetic field; and (ii) a control unit configured to vary said magnetic field over time in order to carry out the method of any of the aspects of the present invention.

[0181] To further explain, considering for example steps (b) and (c) of the method of the first aspect, the control unit causes the magnetic field to induce an oscillating or vibrating motion of the permanent magnet for the duration of step (b) and disables the magnetic field from inducing such motion, for example by turning off the magnetic field, for the duration of step (c).

[0182] Said means for generating a magnetic field are preferably those defined further above in relation to the method of the invention, i.e. an electrical conductor or a permanent magnet.

[0183] The electromagnet is an electrical conductor, preferably at least one coil, more preferably a Helmholtz coil, the opening of which is adapted to receive the vessel.

[0184] When using electromagnets, the control unit is preferably configured to deliver any of the preferred time profiles of electrical current as detailed in relation to the methods of the present invention.

[0185] In a preferred embodiment, said device further comprises or further consists of a container, which is preferably as further defined above and may for example be a single container or an array of containers.

[0186] In a further preferred embodiment, the device further comprises or further consists of at least one permanent magnet and / or magnetic particles, insofar as the device comprises the container, the permanent magnet and / or the particles are preferably located inside the container.

[0187] Permanent magnets and magnetic particles are defined further above.

[0188] Generally speaking, preferred embodiments of one aspect define preferred embodiments of another aspect mutatis mutandis.

[0189] It is understood that the device is configured such that the particles and the magnet are inside the vessel and are preferably exposed to a magnetic field generated by the electromagnet or the further permanent magnet outside the vessel.

[0190] The term "accommodates" means that the coil has an opening that is wide enough for the container to fit inside the opening so that the contents of the container are positioned when the magnetic field generated by the coil in use is particularly strong and / or particularly homogeneous. Preferably, also in the case of containers having a circular cross section (e.g. cylindrical containers), In the case of a vessel, the inner diameter of the coil (in which case it is a circular coil) is only slightly wider than the outer diameter of the vessel. "Slightly wider" can mean 0.01 to 10%, such as 0.1 to 1% wider.

[0191] In the case of an array of vessels (e.g., a microtiter plate), the coil may be only slightly wider than the array. If the array is rectangular in shape, a coil having a rectangular shape, such as a Helmholtz coil, may be used.

[0192] The control unit may further include a power source or adapter to be connected to the electrical plug.

[0193] The device may be provided with a manual containing instructions for carrying out any of the methods of the invention. EXAMPLES

[0194] The examples illustrate the invention.

[0195] Example 1 Performance comparison with prior art magnetic separation devices

[0196] material Saccharomyces cerevisiae cell pellets with a protein content of approximately 100 μg were used for the digestion studies. A permanent neodymium magnet was used for bead manipulation (MagnetExpert; spherical, 2 mm). For lysis and clean-up, the iST-Kit containing buffers and plasticware was used (PreOmics GmbH, PO00001). Trypsin magnetic microparticles were provided by ReSyn Biosciences (ReSyn Biosciences (Pty) Ltd). An external oscillating magnetic field was generated using a Helmholtz coil setup.

[0197] method Traditional magnetic particle manipulation S. cerevisiae cell lysis was prepared as described in standard iST sample preparation (PreOmics GmbH, iST-Kit, PO00001). 750 μg of immobilized trypsin beads (trypsin magnetic microparticles) were equilibrated with 2x 70% ethanol; 1x 1% ammonium hydroxide; 3x 50 mM Tris, pH 8. For each equilibration step, the beads were incubated for 3 min at 500 rpm, the supernatant was removed using a magnetic separator, and 30 s was waited for the magnetic microparticles to collect. The equilibrated beads were mixed with the denatured yeast sample. Digestion was carried out for 1 h at 37°C and 500 rpm. Prior to peptide purification and LC-MS analysis (see standard iST sample preparation), the magnetic trypsin beads were removed from the sample using a magnetic separator and 100 μl of stop buffer was added. After elution, the purified peptides were dried in a SpeedVac and resuspended in 2% acetonitrile, 0.1% trifluoroacetic acid. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled to a Thermo LTQ Orbitrap XL. A peptide load of 5 μg was run on a homemade C 18 Separation on column was followed by tandem mass spectrometry using the DDA Top 10 method. MS / MS data were searched against a yeast database using MaxQuant software with default settings except that a nonspecific search was used.

[0198] Magnetic particle manipulation using a novel system S. cerevisiae cell lysis was prepared as described in standard iST sample preparation (PreOmics GmbH, iST-Kit, PO00001). 750 μg of immobilized trypsin beads (trypsin magnetic microparticles) were equilibrated with 2x 70% ethanol, 1x 1% ammonium hydroxide and 3x 50 mM Tris, pH 8 in a Helmholtz coil setup with a 2 mm circular neodymium magnet. For each equilibration step, the sample was incubated for 3 min in the Helmholtz system applying a 120 Hz square wave function, and the supernatant was removed after the magnetic field was turned off and the magnetic particles were allowed to collect for 10 s. The equilibrated beads were mixed with the denatured yeast sample and digestion was carried out for 60 min in the Helmholtz system applying a 120 Hz square wave function. Prior to peptide purification and LC-MS analysis (see standard iST sample preparation), the magnet and magnetic trypsin beads were removed from the sample and 100 μl of stop buffer was added. After elution, purified peptides were dried in a SpeedVac and resuspended in 2% acetonitrile, 0.1% trifluoroacetic acid. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled to a Thermo LTQ Orbitrap XL. A 5 μg peptide load was separated on a homemade C18 column with a 45 min gradient and subjected to tandem mass spectrometry using the DDA Top 10 method. MS / MS data were searched against a yeast database using MaxQuant software with default settings except using a non-specific search.

[0199] result See Figure 1 and its caption.

[0200] Consideration In terms of the number of peptides identified by MS, the method of the invention performs at least as well as the classical manipulation of magnetic particles, but as discussed herein above, the method of the invention offers distinct advantages, e.g. no mechanical movement of the magnet outside the vessel is required, avoiding any cross-contamination.

[0201] Example 2 material Commercially available Saccharomyces cerevisiae was resuspended in H2O and 1 ml aliquots with an OD at 600 nm of 0.6 were prepared and centrifuged for cell pellets containing approximately 100 μg yeast protein. Standard sample preparation for LC-MS analysis was performed using the iST-Kit (PO00001) from PreOmics GmbH, which includes buffers, enzymes and plasticware. A permanent neodymium magnet was used for bead manipulation (MagnetExpert; spherical, 2 mm). ZrO2 magnetic microparticles were attached to the ReSyn The peptides were supplied by ReSyn Biosciences (ReSyn Biosciences (Pty) Ltd). An external oscillating magnetic field was generated using a Helmholtz coil setup. Peptide cleanup was performed using cation exchange cartridges.

[0202] method For yeast samples, sample preparation was performed according to the PreOmics standard protocol (PreOmics GmbH, PO00001), followed by phosphopeptide enrichment with magnetic microparticles (ReSyn Biosciences) and purification of the samples using reversed-phase cartridges. The purified peptides were thoroughly dried under vacuum at room temperature.

[0203] For enrichment of phosphopeptides, first the magnetic beads were equilibrated. A spherical neodymium magnet with a radius of 2 mm was added to 10 μl of the bead suspension. The suspension was clarified and the supernatant was taken. 50 μl of washing buffer (70% ethanol) was added and the sample was mixed using an external oscillating magnetic field (120 Hz, square waveform, 5 min). Then the suspension was clarified and the supernatant was taken. This procedure was repeated once for a total of two washes with this buffer.

[0204] 50 μl of 1% ammonium hydroxide was added and the sample was mixed again using the method of the present invention (120 Hz, square waveform, 10 min). Again, the sample was clarified and the supernatant was removed. The beads were then equilibrated three times with 50 μl of 0.2 M glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile using the same procedure as above.

[0205] 100 μl of 0.2 M glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile was then added on top of the dried peptide pellet and properly resuspended. The solution was then added to the equilibrated beads and incubated with mixing as per the method of the present invention (120 Hz, square waveform, 20 min). The sample was clarified and the supernatant was taken. Unbound sample was removed by adding another 50 μl of 0.2 M glycolic acid in 5% trifluoroacetic acid in 80% acetonitrile and mixing as per the method of the present invention (120 Hz, square waveform, 2 min). The sample was clarified and the supernatant was taken.

[0206] 100ul of wash buffer (1% trifluoroacetic acid in 80% acetonitrile) was added and the sample was mixed by the method of the present invention (120Hz, square waveform, 2min), after which the sample was clarified and the supernatant was removed. This step was repeated with another wash buffer (0.2% trifluoroacetic acid in 10% acetonitrile).

[0207] Phosphopeptides were eluted from the magnetic beads by adding 40 μl of elution buffer (1% ammonium hydroxide) and incubating with mixing using the method of the present invention (120 Hz, square waveform, 5 min). Samples were clarified and the supernatant was removed and transferred to a new tube. Elution was repeated twice, resulting in a total elution volume of 120 μl.

[0208] The tubes containing the elution were centrifuged at maximum speed to pellet the magnetic bead fragments. The supernatant was removed using a magnetic separator and transferred to a new tube. The samples were dried under vacuum at room temperature until completely dry.

[0209] The cation exchange cartridge was then equilibrated by adding 200 μl of methanol followed by centrifugation in the waste tube (3.800 rcf, 1 min). A second equilibration was performed using 200 μl of 2% acetonitrile, 0.1% trifluoroacetic acid buffer as described above.

[0210] The dried phosphopeptides were resuspended in 200 μl of 2% acetonitrile, 0.1% trifluoroacetic acid buffer, loaded into the cartridge and centrifuged (3.800 rcf, 1 min). The bound peptides were then washed by adding 200 μl of 0.1% formic acid followed by centrifugation (3.800 rcf, 1 min). This washing step was repeated twice. The cartridge was transferred to a new collection tube and the peptides were eluted from the cartridge by adding 200 μl of 0.1% formic acid in 80% acetonitrile followed by centrifugation (3.800 rcf, 1 min). 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 of 2% acetonitrile, 0.1% trifluoroacetic acid buffer.

[0211] Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled to a Thermo LTQ Orbitrap XL. Peptides were analyzed using a homemade C 18 Column separation was followed by tandem mass spectrometry using the DDA Top 10 method. MS / MS data were searched against a yeast database using MaxQuant software with default settings.

[0212] result TIFF2025076424000002.tif19123

[0213] Consideration The specificity evident from the results demonstrates that the method of the present invention works well for separation and enrichment purposes.

[0214] Example 3 Magnetic particle dispersion using various waveforms

[0215] material A permanent neodymium magnet with a parylene coating was used (cylindrical; 2 mm x 2 mm). 3.0 μm amine-functionalized beads were used as magnetic particles. An external oscillating magnetic field was generated using a Helmholtz coil. Online Tone Generator (https: / / onlinetonegenerator.com / ) was used to generate various waveforms at defined frequencies.

[0216] method The magnetic particles were resuspended by mixing thoroughly for 1 min. Three aliquots, each containing 50 μl of particles, were transferred into 0.5 ml screw-cap tubes. The material was washed once with 50 μl of ultrapure water (LC-MS grade; Fisher Scientific) and resuspended in 50 μl of ultrapure water. One magnet was added per tube. The tubes were transferred to a Helmholtz coil and a square wave function, a sine function, a sawtooth function or a triangle function was applied repeatedly for 30 s each at 1–140 Hz. After the magnetic field was turned off, the magnetic particles were allowed to settle on the magnet for 30 s.

[0217] result For all applied waveforms, it was found that the magnetic particles were mixed in the vessel while the magnetic field was on, and when the magnetic field was turned off, the magnetic beads settled onto the magnet in the vessel. Mixed effect differences could be observed (see Table 1).

[0218] [Table 1]

[0219] Consideration Experiments demonstrate that dispersion of magnetic particles can be achieved using a variety of waveforms at different frequencies.

[0220] Example 4 Dispersion of magnetic particles using different coils

[0221] material A permanent neodymium magnet with a parylene coating was used (cylindrical; 2 mm x 2 mm). 3.0 μm amine-functionalized beads were used as magnetic particles. 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 the external oscillating magnetic field. An Online Tone Generator (https: / / onlinetonegenerator.com / ) was used in combination with an amplifier (SMSL SA-50 2x50W) to generate various waveforms at defined frequencies.

[0222] method The magnetic particles were resuspended by mixing thoroughly for 1 min. Three aliquots, each containing 50 μl of particles, were transferred into 1.5 ml Eppendorf reaction vessels. The material was washed once with 50 μl of ultrapure water (LC-MS grade; Fisher Scientific) and resuspended in 50 μl of ultrapure water. One magnet was added per tube.

[0223] The reaction vessels were placed in the coil setup (one vessel per coil). The samples were incubated while a square wave function, a sine function, a sawtooth function or a triangle function was applied repeatedly at 1-180 Hz for 30 seconds. After the magnetic field was turned off, the magnetic particles were allowed to settle on the magnet for 10-30 seconds.

[0224] result It was found that the magnetic particles mixed in the vessel in a single coil setup while the magnetic field was on, and when the magnetic field was turned off, the magnetic beads settled onto the magnet in the vessel. Differences in the mixing effects at different frequencies could be observed (see Table 2).

[0225] [Table 2]

[0226] Consideration Experiments have shown that dispersion of magnetic particles can be achieved in various frequency ranges and with various waveforms using a simple coil set-up, ie, without the need for Helmholtz coils.

Claims

1. 1. A method for dispersing magnetic particles, comprising the steps of: (a) combining in a vessel at least one permanent magnet and the magnetic particles in a liquid phase; (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field; wherein said particles are dispersed by means of said dispersion means.

2. The magnetic particle has at least one moiety on its surface, the moiety comprising: (i) a moiety capable of binding to a target molecule, preferably a binding protein, an affinity chromatography material, an absorbent material, an adsorbent material, a probe, or a primer; (ii) a moiety capable of converting at least one initiator molecule into at least one product molecule, the moiety being preferably an enzyme or chemical catalyst; and (iii) a moiety capable of forming an adduct with a target molecule and the target molecule or initiating molecule, respectively, is present or suspected to be present in the liquid phase.

3. (c) removing, staticating and / or disabling the magnetic field from inducing the movement and concentrating the magnetic particles on the permanent magnet; (d) removing the liquid phase; 3. The method of claim 1 or 2, further comprising or consisting of:

4. 1. A method for separating a compound from a mixture, comprising the steps of: (a) contacting the mixture in a container with magnetic particles having moieties capable of binding to the compound and at least one permanent magnet; (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field; by which said compound is separated from the rest of the constituents of said mixture.

5. (c) removing, staticating and / or disabling the magnetic field from inducing the movement and concentrating the magnetic particles on the permanent magnet; (d) removing the remainder of the components of the mixture from the container; and 5. The method of claim 4 further comprising or consisting of:

6. (e) adding a cleaning solution to the container; (f) inducing an oscillating or vibrating motion of the permanent magnet using the magnetic field; (g) removing, staticating, and / or disabling the magnetic field from inducing the movement, and concentrating the magnetic particles on the permanent magnet; (h) removing the cleaning solution from the container; The method according to claim 4 or 5, further comprising or consisting of: and optionally repeating steps (e) to (h).

7. (i) adding to the vessel an eluent that reduces or destroys the binding of the compound to the moiety; (j) inducing an oscillating or vibrating motion of the permanent magnet using the magnetic field; (k) removing, staticating and / or disabling said magnetic field from inducing said motion and concentrating said magnetic particles on said permanent magnet; and preferably further comprising or consisting of (l) separating the magnetic particles and the resulting eluate from the permanent magnet.

7. The method of claim 5 or 6, further comprising or consisting of:

8. 1. A method for producing at least one product molecule, comprising: (a) contacting in a vessel at least one initiator molecule with magnetic particles having a moiety capable of converting the at least one initiator molecule into at least one product molecule and at least one permanent magnet; (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field; wherein said at least one product molecule is obtained.

9. (c) removing, staticating and / or disabling the magnetic field from inducing the movement and concentrating the magnetic particles on the permanent magnet; (d) removing the at least one product molecule from the vessel; and 9. The method of claim 8 further comprising or consisting of:

10. 1. A method of derivatizing a first compound, comprising the steps of: (a) contacting the first compound in a container with magnetic particles having moieties capable of forming an adduct with the first compound and at least one permanent magnet; (b) inducing an oscillating or vibrating motion of said permanent magnet using a magnetic field; 5. A process for obtaining said adduct, comprising or consisting of:

11. (c) removing, staticating and / or disabling the magnetic field from inducing the movement and concentrating the magnetic particles on the permanent magnet; (d) removing material not bound to the particles from the container; and 11. The method of claim 10 further comprising or consisting of:

12. The magnetic field is generated by an external magnet, the external magnet preferably comprising: (1) an electromagnet, in which a fluctuating or oscillating current passes through the electromagnet; and / or (2) a further permanent magnet or electromagnet, wherein the vessel and the further permanent magnet or electromagnet are moved relative to one another in an oscillating or vibrating manner; 2. The method of any one of the preceding claims, wherein

13. 1. Use of at least one permanent magnet, magnetic particles, and a magnetic field for mixing a reaction mixture in a vessel, wherein said at least one permanent magnet and said particles are within said vessel, and preferably said magnetic field is generated by a further magnet external to said vessel.

14. (i) magnetic particles; (ii) at least one permanent magnet; and (iii) at least one liquid reagent, e.g., a buffer solution; A kit comprising or consisting of:

15. (i) a means for generating a magnetic field; and (ii) a control unit configured to vary said magnetic field over time in order to carry out the method of any one of the aspects of the present invention. A device comprising or consisting of:

16. The means is configured to receive the container such that, in use, the container is exposed to the magnetic field. The device of claim 15 .

17. 17. The device of claim 16, further comprising or consisting of the container.

18. 18. The device of claim 17, further comprising or consisting of a permanent magnet and / or magnetic particles within the container.

Images