Patent Description:
The invention relates to a system for capturing target components from a fluid, for example supra-Brownian reaction chamber systems to enhance molecular collisions in flowing liquids, and more specifically to using self-assembling functionalized magnetic particulate chains which are actuated relative to the target containing fluid in order to increase collision rates between targets and one or more ligands attached to the particulate chain.

Magnetic nanoparticles and micron-sized composite particles have properties useful for the purpose of magnetic separation of target moieties from a fluid. Their surfaces may be functionalized to make them suitable for use as a target-specific binding surface. As a result of this property, magnetic nanoparticles and micron-sized microbeads have been used in laboratory bench-top systems for the purpose of magnetic separation of target components. In such bench-top systems, functionalized magnetic microbeads are mixed with small volumes of a fluid containing both the target moieties and other species in a container. The mixture is then exposed to a permanent magnet that produces an inhomogeneous magnetic field over the entire volume of the container. This actuates and concentrates the magnetic nanoparticles or microbeads at a predetermined location in the fluid container. The non-actuated materials in the supernatant may be separated off, and a chemical or other agent added to the mixture to cleave the bonds between the target moiety and the magnetic entities, thereby allowing the target components to be separated out and then further purified.

Such a magnetic separation method is not suitable if one wishes to apply the magnetic separation to a large volume of a given fluid. Further, the time required for the Brownian motion-mediated separation process when the container is placed near the magnet may be too long and thereby damage the components in the fluid.

What is needed is a method of accelerating the binding time between functionalized magnetic particles and target moieties.

The present invention addresses these requirements. <CIT> discusses magnetic mixing in micron size droplets. <CIT> describes manipulating and mixing magnetic particles in a liquid medium.

The invention provides a system for capturing target components from a liquid as set out in claims <NUM> and <NUM>, and a method as set out in claim <NUM>.

Embodiments of the invention relate to a molecular magnetic tagging system which creates a high collision rate between magnetic particles and target moieties in a flowing fluid. A fluid containing the magnetic particles is flowed through a magnetic field of controlled magnitude rotating at a controlled rate in such a way that the magnetic particles form rotating chains of a tunable length. These chains serve to increase the collision rate between the magnetic particles and their target moieties, thus decreasing the overall binding time.

In one embodiment, the system comprises a fluid including a plurality of magnetic particles that flows from input port to output port. In another embodiment, the fluid further comprises a plurality of magnetic nanoparticles. In yet another embodiment, the magnetic particles comprise a ligand. In still yet another embodiment, at least one of the magnetic particles and the magnetic nanoparticles comprises a ligand. In another embodiment, the molecular mixing system includes a motorized turntable; a speed controller to control the rotational velocity of the motorized turntable; a plurality of magnets arranged in a first Halbach array, the first Halbach array located on the motorized turntable and concentric to the axis of the motorized turntable; and a sample conduit having an input port and an output port and having an outer wall defining a lumen, the sample conduit positioned within the first Halbach array. In yet another embodiment, in operation, the first Halbach array rotates circumferentially about the outer circumference of the sample conduit.

In one embodiment, the sample conduit comprises a sample container. In another embodiment, the sample container includes an inner wall concentric with the outer wall and defining a lumen between the inner and outer wall, the lumen in fluid communication with the input and output ports. In yet another embodiment, the system comprises a second Halbach array of magnets within the inner wall of the sample container. In still yet another embodiment, the first Halbach array has a K = <NUM>. In another embodiment, the second Halbach array has a K = <NUM>. In yet another embodiment, the sample container has a truncated conical shape with the largest portion of the cone at the input port and the narrowest portion of the cone at the output port. In still another embodiment, the Halbach array comprises a plurality of Halbach arrays stacked axially adjacent one another. In still yet another embodiment, the plurality of stacked Halbach arrays are separated by a space. In still yet another embodiment, the plurality of Halbach arrays have varying heights and are stacked from the greatest height to the smallest height from input port to output port, through which the fluid could flow coaxially in either direction. In another embodiment, the molecular mixing system includes: a sample conduit having an input port and an output port and having an outer wall defining a lumen; a motorized carrier; a speed controller to control the rotational speed of the motorized carrier; and a pair of electromagnets attached to the motorized carrier and positioned on opposite sides of the sample conduit, wherein in operation the pair of electromagnets rotates circumferentially about the outer circumference of the sample conduit.

Embodiments of the invention also relate to a method of mixing at a molecular level. In one embodiment, the method includes: providing a sample conduit having an input port and an output port; passing a fluid comprising a plurality of magnetic particles through the sample conduit from input port to output port; applying a magnetic field through the sample conduit; and rotating the magnetic field about an axis coaxial with the longitudinal axis of the sample conduit with an angular velocity. In another embodiment, the method further includes the step of adjusting the strength of the magnetic field and the angular velocity of the rotation of the magnetic field so as to form chains of magnetic particles.

This disclosure also relates to a method of forming magnetic chains of selectable numbers of particles, for example a method comprising passing the fluid containing the magnetic particles through a magnetic field of controlled magnitude rotating at a controlled rate such that the magnetic particles form rotating chains of a selectable size.

The structure and function of the invention can be best understood from the description herein in conjunction with the accompanying figures. The figures are not necessarily to scale, emphasis instead generally being placed upon illustrative principles. The figures are to be considered illustrative in all aspects and are not intended to limit the invention, the scope of which is defined only by the claims.

In brief overview and referring to <FIG>, an embodiment of a system constructed in accordance with the invention includes a tagging chamber <NUM> that includes a magnetic array <NUM> through which a sample chamber or conduit <NUM> is positioned. The magnetic array is located on a turntable <NUM> controlled by a controller <NUM>. The turntable may be rotated by a standard motor or a stepper motor. A sample of interest <NUM> is pumped by a suitable pump <NUM> (a syringe pump is shown for clarity, but any pump may be used) through the input port <NUM> of the sample chamber or conduit <NUM> to the output port <NUM> of the sample chamber or conduit <NUM> for additional processing. A second pump <NUM> with a solution of magnetic separation particles <NUM> is also connected to the input port <NUM> of the sample conduit or chamber <NUM> through a T-connector <NUM> and mixes the magnetic separation particles <NUM> with the sample <NUM>.

Once the sample <NUM> and the magnetic separation particles <NUM> have been mixed magnetically in the sample conduit or chamber <NUM>, the mixture may be further treated as appropriate for the sample. In the embodiment shown, the mixture is passed to a High Gradient Magnetic Separation (HGMS) device <NUM> that separates targeted cells <NUM> from the other components <NUM> of a blood sample <NUM>.

In operation, the magnetic separation particles <NUM> may be of two forms. In the first form, the magnetic particles are not small enough to be considered nanoparticles. Each particle, which in one embodiment is an iron oxide, is ferromagnetic and so is not magnetic in the absence of a magnetic field. This is because the magnetic domains of the particles are randomly oriented when no magnetic field is present. The particles therefore exist as individual particles in solution. In one embodiment, each individual magnetic particle is bound to a ligand that is unique to the component of interest in the sample. When the component in the sample is captured by the ligand of the magnetic particle, the component of interest is bound to the magnetic particle by the ligand.

When exposed to a magnetic field, the magnetic domains of the magnetic particle stop being randomly magnetized and align, and the magnetic particles develop a magnetic dipole. The magnetic particles then stick to other magnetic particles and form magnetic particle chains. The chains of particles are fairly stable and will remain as chains in a solution as long as any external forces applied to the chain do not overcome the dipole magnetic forces holding the chain together.

The magnetic array <NUM> is constructed to rotate with the turntable <NUM>, thereby causing the magnetic field traversing the sample conduit or chamber <NUM> to rotate as discussed below. As the substantially homogeneous magnetic field rotates, the magnetic particle chains respond to the rotation of the magnetic field and begin to rotate also so as to align with the magnetic field. As a chain rotates, the chain experiences two forces: the force caused by the magnetic field, and a resistive drag force such as the Stokes drag force, which arises because the chain is moving through a viscous medium, the solution. The Stokes force increases as the rotational velocity of the rotating magnetic particle chain increases. As the rotational velocity of the magnetic field increases beyond a value determined by the rotational velocity of the chain, the length of the chain, and the viscosity of the solution, the Stokes force generated by the movement of the chain through the solution overcomes the dipole magnetic force holding the chain together and the chain begins to fragment or divide into more chains.

However, if the rotational velocity of the magnetic field is maintained by the controller <NUM> at a velocity less than what is necessary to fragment the entire chain, the portion of the chain remaining will rotate without further fragmentation. As the chain rotates, the particles that make up the chain sweep a volume space and any target component of interest that is in the swept volume will be captured by the ligands bound to the magnetic particles. This increases the number of collisions per unit time between the magnetic particles and the components of interest, thus increasing the binding efficiency and subsequent capture process.

In these embodiments, the motor rotating the turntable can be a standard AC or DC electric motor or it can be a stepper motor. In the case of an AC or DC motor, the controller <NUM> can use a simple voltage drop across a rheostat to control the speed of the motor and hence the turntable. In the case of a stepper motor, a pulse controller <NUM> is used to send pulses to the stepper motor at various repetition rates to control the rotational speed.

Coating magnetic particles is relatively expensive for various ligands, such as antibodies. To avoid this expense, a second embodiment of magnetic particles is used. In this embodiment, magnetic nanoparticles are added to the magnetic particles in solution. Rather than coating the larger magnetic particles with the ligand, the magnetic nanoparticles are coated with the ligand. Because the magnetic nanoparticles have a smaller surface area, less ligand is needed and the cost of the coated particles is less. When the magnetic nanoparticles and the magnetic particles are exposed to the magnetic field, the magnetic particles again form chains and the magnetic nanoparticles collect at the junctions of the chains of magnetic particles (<FIG>). As the magnetic particle chains rotate, the magnetic nanoparticles rotate with the chains, again sweeping a volume of sample and capturing components that are of interest.

In more detail, in one embodiment, the sample conduit or chamber <NUM> is simply a tube through which the sample solution passes. In another embodiment, and referring to <FIG>, the sample conduit or chamber <NUM> is a chamber having the structure shown in <FIG>, constructed of polyethylene. In this embodiment, the sample chamber has an input port <NUM> at one end of a cylindrical container <NUM>. At the other end of the cylindrical sample chamber is an output port <NUM>. As with the previous conduit embodiment, the sample chamber <NUM> is sized to fit within the magnetic array <NUM>.

The sample with the component of interest <NUM> and the magnetic particles <NUM> merge in the input port and enter the chamber <NUM>. Once in the chamber <NUM>, the magnetic field from the array <NUM> causes the magnetic particle chain formation and rotation, dispersed evenly across the chamber cross-section. As fluid leaves the chamber <NUM> through the output port <NUM>, the chains of magnetic particles and bound target components are swept toward the output port <NUM>. A cone <NUM> is placed along the axis of the input and output ports <NUM> and <NUM> respectively to deflect the flow toward the walls of the chamber <NUM>, increasing the flow velocity around the cone in order to prevent clogging of the system at the exit. As will be discussed below, the field generated by the magnetic array <NUM> increases in strength toward the direction of the input port <NUM> near the chamber wall, the positive magnetic gradient creating a magnetic force which drags the magnetic particle chains with the fluid. This moves the magnetic particle chains away from the output port <NUM> and delays the elution of the magnetic particle chains from the chamber <NUM> by way of the output port <NUM>. This increases the amount of time the ligands have to encounter a target component because the chains exist for longer within the chamber.

Referring to <FIG>, in another embodiment of the chamber <NUM> the central volume of the chamber is replaced with a cylinder <NUM> so as to form an elongated toroidal space <NUM>. <FIG> depicts the chamber <NUM> with one end removed to show the creation of the toroidal volume <NUM> by the wall of the chamber <NUM> and the wall of the inner cylinder <NUM>. <FIG> shows the fluid flow from the input port <NUM> around the cylinder <NUM> to the output port <NUM>.

Referring now to <FIG>, another embodiment of the conduit <NUM> is depicted as a truncated cone <NUM>' within the magnetic array <NUM>. In this embodiment, as the sample <NUM> and the magnetic particles <NUM> move from input port <NUM> to output port <NUM> of the conduit <NUM>', the available volume decreases due to the decreasing diameter of the conduit <NUM>'.

Referring to <FIG>, in one embodiment the magnetic array is constructed as a single Halbach array having a plurality of permanent magnets such as neodymium-iron-boron magnets. In this embodiment, there are twelve permanent magnets (<NUM>, <NUM><NUM>, <NUM><NUM>,. ,<NUM><NUM>, generally <NUM>) arranged in a cylinder. At both ends of the cylinder are two magnetic rings <NUM>, <NUM>' to increase the magnetic field at the ends of the array so as to maintain the homogeneity of the magnetic field along the device. <FIG> depicts the Halbach array with an external circumferential nonmagnetic support <NUM> to maintain the permanent magnets <NUM> in their proper positions. In various embodiments, the support may be made from, but not limited to, aluminum, plastic or wood.

Referring to <FIG>, a Halbach array produces one or more magnetic fields inside the array depending on the orientation of the magnetic dipole of the individual permanent magnets that make up the array. Referring to <FIG>, in this embodiment the twelve permanent magnets are arranged with their dipoles oriented outward from the array. This results in no field being generated within the array.

In order to quantify the various types of fields generated by the array, one can count how many revolutions of the dipole take place as one starts with one dipole and moves around the circumference of the array. In <FIG>, starting at the <NUM>:<NUM> o'clock position and moving around the array clockwise, one sees that the dipole at the <NUM>:<NUM> o'clock is oriented in the same direction as the dipole at <NUM>:<NUM> o'clock. Continuing clockwise around the array, the dipole again rotates back to the same position at <NUM>:<NUM> o'clock. Thus, the dipole has rotated twice as we look at the orientation of the dipole as we move once around the array. This array then has a "K" value of <NUM>. This array also produces a magnetic field vector pointed in one direction as indicated by the arrow within the array.

In the previous embodiment, the dipole rotated only once as we moved about the array, so it had a K value of "<NUM>". In <FIG>, k= <NUM> and in <FIG>, k=<NUM> and the magnetic field within the array takes on complex forms. The K value of the embodiment of the Halbach array as used herein is "<NUM>".

A chamber or conduit <NUM> placed within the Halbach array of K=<NUM> will experience a significant magnetic field across a diameter of the conduit or chamber <NUM>. <FIG> is a graphical representation of the result of a mathematical model of a K=<NUM> magnetic field across a segment of a conduit or chamber <NUM> oriented perpendicular to the longitudinal axis of the chamber or conduit <NUM>. It is apparent that in the orientation shown, the magnetic field axis is primarily along one axis of the graph. If the Halbach array is then rotated about its longitudinal axis, the magnetic field across the array will also rotate and a magnetic particle chain positioned within the chamber or conduit <NUM> within the array will experience the rotating magnetic field and rotate also.

As the chain is dragged by the fluid passing through the conduit or chamber <NUM> from the input port <NUM> to the output port <NUM>, it encounters an additional field near the walls of the conduit or cylinder <NUM> directed against the flow of the fluid, carrying the magnetic chain back into the body of the chamber <NUM>. <FIG> is a graphical representation of the K=<NUM> magnetic field along the longitudinal axis of conduit or chamber <NUM> at the output port-end of the chamber or conduit <NUM>. As the arrows indicate, a magnetic particle chain will experience a force toward the walls of the chamber or conduit <NUM> followed by a downward flow back into the chamber or conduit <NUM>. This will cause the magnetic particle chain to remain longer in the chamber or conduit <NUM> to interact with the target component of interest. The cone <NUM> shown in <FIG> is constructed of a plastic such as polycarbonate and aids in moving the magnetic particle chains toward the walls of the chamber <NUM>, so that each magnetic particle chain will encounter the magnetic field directing the particle chain back into the chamber.

The Halbach array can be modified to accommodate various forms of chamber. Referring to <FIG>, this embodiment of the Halbach array is constructed to accommodate the chamber of <FIG>. An inner Halbach array <NUM> fits into the inner space formed by the inner cylinder <NUM>. In the array, the magnetic field vectors (<FIG>) produced by the inner and outer Halbach arrays sweep through the sample portion of the chamber, allowing the reduced space of confinement of the magnetic chains and the target components to experience a moving magnetic field as in the other embodiments.

<FIG> is perspective view of another embodiment of the Halbach array. In this embodiment, a plurality of Halbach subarrays are stacked around a chamber or conduit <NUM>. In this embodiment, each subarray is of a different height (h) with a space <NUM> between each subarray. The embodiment shown is three subarrays in height, but other stacked arrays are contemplated.

Referring to <FIG>, the device can also be constructed by placing solenoid electromagnets <NUM>, <NUM>' on opposite sides of the conduit or container <NUM>, the magnets oriented with their poles having opposite polarity. In this way, a magnetic field F passes from one magnet to the other through the container <NUM>. The electromagnets <NUM>, <NUM>' are mounted on the turntable <NUM> and rotate about the conduit or container <NUM>.

A discussion of the physics behind the operation of the system begins by considering that the Stokes drag force (Fd) on a spherical particle moving through a fluid is given by the equation: <MAT>.

The maximum length of the chain occurs when the drag force equals the magnetic force between the magnetic particles: <MAT>.

At this point, the force is such that the particles at the ends of the chain begin to fragment off the chain.

The length of the particle chain under rotation is then given by: <MAT>.

Chains of magnetic particles sweep the target components from the liquid more effectively than single particles. To understand this, assume that the probability of a collision between a target component and a magnetic particle is a function of the area occupied by the magnetic particle. If a target component with diameter (D) is moving through the liquid, a region of interaction of the target component with the magnetic particles, each having a radius (R), will be a slice through the liquid with an interaction thickness of (2R)+D. The density of the magnetic particles per slice is (ρ). If there are no chains of magnetic particles, i.e. monodisperse particles, the total area occupied by the magnetic particles is ρπR<NUM>.

If chains of magnetic particles are formed, the number of magnetic particle chains per ml. of liquid is: 2ρR/l, where the number of chains per ml equals number of particles (ρ) per ml times the diameter (<NUM>R) of each particle divided by length (l) of each chain.

Time for the target component to travel through slice is: t=(D+2R)/v, where (v) is the velocity of the target component and depends on flow rate and dimensions of chamber/tubing/cylinder. If t > <NUM>/2ω, the rotating chain will complete more than half a rotation which is equivalent to covering the whole area of the circle, so the area covered by the chains is the number of chains (2pR/l) times the area swept (π(l/<NUM>)<NUM>): <MAT>.

If this is compared to the area covered by the monodisperse particles (ρπR<NUM>), one sees: <MAT>.

As a result, if (l > 2R), the chains sweep and occupy more area than a group of single particles. Because (2R) is the diameter of a single particle, all chains will be better at capturing target components if the transit time (t) is greater than (<NUM>/2ω). This is also shown diagrammatically in <FIG>, in which the same number of particles cover a greater area when acting as rotating chains (B) than as individual particles of the same size (A).

If the velocity of the target component is increased such that the transit time t < <NUM>/(2ω), then the magnetic chains will not complete a full rotation and the area swept by the rotating chains will be less. The area covered by the rotating chains is: <MAT>.

This area is larger than the area of monodisperse particles: <MAT> only if: <MAT>.

Remembering that: <MAT> <MAT> <MAT>
also remembering that: <MAT>
then: <MAT>.

As a result, chains are only better if the velocity of the target component (v) is: <MAT>.

That is, the flow rate should be low enough that the chains complete a full rotation.

The limit on the rotational frequency of the field for a particular particle may be calculated as: <MAT> <MAT> <MAT>.

Remembering: <MAT> and <MAT> <MAT> <MAT>.

This last equation gives the maximum rotational frequency beyond which chains no longer form. Alternatively, if the magnetic field strength is controlled such that the magnetic particles are not saturated, the relationship becomes: <MAT>.

The actual difference made by an increase in rotational frequency (ω) may be calculated assuming that the number of collisions between the target component and the magnetic particles is directly related to the area covered by particles such that as the area is doubled, so are the collisions. The total area of monodisperse particles is: <MAT> and the total area of rotating magnetic particle chains at full rotation is: <MAT>.

The more omega is increased beyond a minimum, the smaller the improvement due to formation of chains.

Unless otherwise indicated, all numbers expressing lengths, widths, depths, or other dimensions and so forth used in the specification and claims are to be understood in all instances as indicating both the exact values as shown and as being modified by the term "about. " Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Any specific value may vary by <NUM>%.

The terms "a," "an," "the," and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of any claim. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments are described herein, including the best mode known to the inventor for carrying out the present disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventor intends for the invention to be practiced otherwise than specifically described herein. Moreover, any combination of the above-described elements in all possible variations thereof is contemplated unless otherwise indicated herein or otherwise clearly contradicted by context.

Claim 1:
A system for capturing target components of interest from a liquid, comprising:
a motorized turntable (<NUM>);
a speed controller (<NUM>) to control the rotational velocity of the motorized turntable (<NUM>);
a plurality of magnets (<NUM>) arranged in a first Halbach array, the first Halbach array located on the motorized turntable (<NUM>) and concentric with the axis of the motorized turntable so as to rotate circumferentially about the outer circumference of a sample conduit;
the sample conduit (<NUM>) having an input port (<NUM>) and an output port (<NUM>) and having an outer wall defining a lumen, the sample conduit positioned within and concentric with the axis of the first Halbach array; and
a liquid (<NUM>) containing a plurality of magnetic particles (<NUM>) flowing through the sample conduit from the input port to the output port, wherein each individual magnetic particle (<NUM>) is bound to a ligand that is unique to the target components of interest in the sample,
wherein, in operation, the magnetic field of the first Halbach array has a strength and an angular velocity of rotation adjusted such that the magnetic particles (<NUM>) form chains of magnetic particles within the liquid (<NUM>), so that the target components of interest are captured by the ligands bound to the magnetic particles (<NUM>).