Device and method for mixing and bubble removal

A magnetic mixing device designed to mix fluid in a reaction chamber and remove air bubbles if present. The device comprises a holder with embedded magnets, which causes movement of a stir bar within the reaction chamber. The holder may be moved by an electric linear actuator configured to generate linear motion or an electric motor configured to generate a circular motion. When orientated so the stir bar moves vertically within the reaction chamber, the stir bar disrupts any air bubbles trapped within or below the fluid.

BACKGROUND

Technical Field

The present disclosure relates to the field of mixing devices and mixing methods, and in particular, to a magnetic mixing device and method for using a magnetic spin bar to circulate fluid and remove air bubbles within a reaction chamber.

Background

The mixing of solutions is routinely used in many industrial processes and is often essential in some chemical and biological reactions. Mixing is beneficial in any chemical or biological reaction where an equal and homogeneous concentration is needed throughout the solution.

In biotechnology, mixing is generally performed by methods such as pipette mixing or vortexing. There are advantages and disadvantages of each method, for example, mixing may yield variability between samples.

Biotechnology companies use extensive amounts of culture media, buffers, and reagents. Such materials originally come in powdered form and must be rehydrated prior to use. Rehydrating the reaction components reduces reaction time and improves consistency between reactions.

In one area of biotechnology where rehydration of reaction components is done for nucleic acid amplification, the reaction components may be rehydrated by pipette mixing or vortexing, or thermal mixing before the reaction begins. Pipette mixing requires both specialized laboratory skills and specialized laboratory equipment. Vortexing requires specialized laboratory equipment that is not typically battery operated or portable. Both vortexing and pipette mixing are variable, depending on the manner in which the operator performs the process. For example, there may be variability in the number of times the fluid is cycled through the micropipette or the duration of vortexing. Thermal mixing occurs during the temperature cycling, however optimal amplification may be delayed a few cycles until the solution is properly mixed. Thermal mixing is time-consuming, often taking approximately 10 minutes for the reaction solution to become thoroughly mixed. When using the invention, all parameters of the process are controlled, for example motor ramp up rate, motor revolutions per minute, motor duration at maximum revolutions per minute, and motor ramp down rate.

Air bubble(s) may get trapped within or beneath the fluid. Traditionally, the air bubble is removed by methods such as a centrifugation or pipette mixing. Another common method of removing air bubbles is tapping the reaction plate or the tube. In a magnetic mixer where the stir bar rotates at the bottom of the reaction chamber, the air bubble may not be disrupted and may remain at the bottom of the reaction chamber. An air bubble can cause inconsistent results for a number of reasons including reducing the effective volume of the reaction, preventing the reaction from achieving the appropriate reaction temperatures, interfering with a detector, and preventing the complete mixing of reaction components.

A device that quickly rehydrates dried reaction components, produces evenly distributed mixing throughout the reaction volume, and removes air bubbles in a reaction chamber is highly desirable. It is also advantageous if this device is designed so any operator can use it, so it does not require a trained technician. Therefore, it is desirable that the device be configured to alert a user if the device is not operating properly.

SUMMARY

The present disclosure is directed to a magnetic mixer that can be used for mixing in any chemical or biological application. It is to be understood that the term “mix” in this disclosure refers to any movement that creates a uniform solution, e.g. mix, stir, blend, agitate, etc.

It is to be understood that the term “holder” in this disclosure refers to any mechanical expedient, e.g. support, spindle, bracket, prop, etc.

Consistent with a disclosed embodiment, a device is disclosed that quickly mixes solutions and removes air bubbles that may be present in the reaction chamber. One application of the device and method of the present disclosure is the quick rehydration of dried reaction components by a magnetic mixer. In a nucleic acid amplification assay, for example, the dried down reaction components must be rehydrated to re-suspend reagents, reduce reaction time and improve consistency between reactions. Dried down reaction components may include, but are not limited to, polymerase chain reaction (PCR) primers, PCR probes, nucleotides, taq polymerase, magnesium chloride, Bovine Serum Albumin, trehalose, and PCR buffer. Dried reaction components may also include, but not be limited to, NASBA, RPA, HDA, LAMP, RCA, ICAN, SMART, SDA, and LDR reaction components.

The geometry of a reaction chamber could cause air bubble(s) to be trapped within or beneath the fluid. The present disclosure describes devices and methods that address both challenges of quick mixing and removing trapped air bubbles.

Consistent with an exemplary embodiment of the present disclosure, a device is disclosed that detects when a magnetic stir bar is moving within a reaction chamber. A control unit detects a change in current to a motor due to the presence of a magnetic stir bar and a feedback loop provides data to a magnetic mixing device or a master instrument. A lack of change in current to a motor represents the absence of a magnetic stir bar within a reaction chamber. The feedback loop to a master instrument can alert an untrained user to the absence of the magnetic stir bar and any other potential problems with the magnetic mixing device.

Additionally, a device is disclosed that contains at least two magnetic stir bars of a shape that promotes a grinding-type action to breakdown a sample.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments and together with the description, serve to explain the principles of the various aspects of the embodiments. Other embodiments of this disclosure are disclosed in the accompanying drawings, description, and claims. Thus, this summary is exemplary only, and is not to be considered restrictive.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present disclosure describes a magnetic mixing device. Exemplary embodiments may include a rare earth magnet that attracts and/or repels a paramagnetic stir bar.

The present disclosure further describes a device that removes trapped air bubbles beneath or within a fluid.

FIG. 1illustrates an exemplary magnetic mixing device10comprising a source of motive power20, a holder30containing an embedded magnet40, a reaction chamber50, stir bar(s)60, housed in an outer housing70. The stir bar(s)60is co-located within the reaction chamber50.

In some embodiments, source of motive power20may be an electric motor, which in an illustrative embodiment is configured to generate a circular motion. In alternative embodiments, source of motive power20may be an electric linear actuator configured to generate linear motion. In other embodiments, the movement of magnet40may be performed without a motor or a linear actuator, for example, by hand or other manual means. The source of motive power20can also be mixed-mode, relying on more than one motor, actuator, manual motion, etc.

Another aspect of the present disclosure is holder30that contains one or more embedded magnet40. In an exemplary embodiment, holder30is embedded with one rare earth magnet40. In alternative embodiments, holder30has more than one rare earth magnet40embedded in holder30. In alternative embodiments, magnet40is another type of permanent magnet. In alternative embodiments, magnet40is an electromagnet. In alternative embodiments, holder30itself is a permanent magnet or an electromagnet. In alternative embodiments, magnet40is moved relative to reaction chamber50without being embedded in a holder.

Reaction chamber50may be configured for various functionalities. In an exemplary embodiment, reaction chamber50may be optimized to perform a nucleic acid amplification reaction. In another exemplary embodiment, reaction chamber50may be contained within cartridge90.

Another aspect of the present disclosure is one or more magnetic stir bar60. The motion of stir bar60is driven by the movement of magnet40. Exemplary shapes of stir bar60may include disc, rod, cross, ring, and any other shape or construction capable of mixing. The size of stir bar60should be coupled to the size of reaction chamber50to allow stir bar60to move unrestricted.

In exemplary embodiments, stir bar60may be stainless steel, though stir bar60may be any magnetic or paramagnetic material. The magnetic material of stir bar60may be coated or uncoated. In exemplary embodiments, stir bar60is coated with a material that does not react with or contaminate the reaction components. In an exemplary embodiment, stir bar60may be coated with parylene. In alternative embodiments, the coating may be any number of coatings other than parylene. In such embodiments, a preferred coating is one that has been FDA approved for use in food, drug, and cosmetic applications.

Another aspect of the present disclosure is housing70, which is used to align source of motive power20and reaction chamber50.

Holder30orientation relative to reaction chamber50may be in any manner that allows for movement of stir bar60within reaction chamber50. In one embodiment, holder30is rotated parallel relative to reaction chamber50. In another embodiment, holder30is rotated perpendicularly relative to reaction chamber50, along the longitudinal axis of reaction chamber50. In another embodiment, mixing device10is configured to allow reaction chamber50to sit at the center and holder30moves magnet40around reaction chamber50. In an exemplary embodiment, holder30may be orientated to rotate alongside of reaction chamber50in a manner so that stir bar60moves vertically within reaction chamber50.

FIG. 2Aillustrates the introduction of dried components110and fluid100within a reaction chamber50.FIG. 2Billustrates the resulting uniform solution120after mixing in a magnetic mixing device of the present disclosure.

An air bubble80may be trapped at the bottom of reaction chamber50when fluid is introduced, as shown inFIG. 1andFIG. 3A. In an orientation where stir bar60rotates at the bottom of reaction chamber50, trapped air bubble80may not be removed by motion of stir bar60. In an orientation that moves stir bar60vertically within reaction chamber50, air bubble80trapped at the bottom of a narrow reaction chamber50can be disrupted and/or air bubble80can be forced to the top of reaction chamber50.

FIG. 3Aillustrates air bubble80trapped in a solution caused by the introduction of a liquid, represented by hatched lines, to reaction chamber50within cartridge90.FIG. 3Billustrates the removal of the air bubble after mixing using a magnetic mixing device of the present disclosure.

In an embodiment where the fluid has a viscosity high enough that air bubbles80may be trapped within the fluid, such as a gel or cream, an orientation that moves stir bar60vertically within reaction chamber50may similarly disrupt air bubbles80.

Further, an orientation that moves stir bar60vertically within reaction chamber50may result in a turbulent flow, rather than a predictable vortex. The vertical motion advantageously creates an evenly distributed mixing throughout the reaction volume.

It will be apparent to those skilled in the art that in embodiments where stir bar60is of a paramagnetic material, the distance between magnet40and stir bar60must be within range to effectively attract or repel stir bar60to cause the desired movement of stir bar60within reaction chamber50.

In an embodiment where there is more than one reaction chamber50, corresponding stir bar(s)60must be in each reaction chamber50. They must be positioned adjacent to magnet40within a range to effectively attract or repel stir bars60to cause the desired movement within respective reaction chambers50.

In an alternative embodiment, magnet40may be stationary without the use of a holder, and reaction chamber50may be moved relative to magnet40by source of motive power20in the form of an electric motor or other manual means.

The present disclosure may contain more than one magnetic stir bar60, within reaction chamber50, of a shape to optimize a grinding-type action to break down a sample.

In an alternative embodiment, a microprocessor controls the ramp rate and speed of the source of motive power20, for instance a motor, and therefore stir bar60. In an exemplary embodiment a master control unit controls magnetic mixing device10and selects a mixing protocol. The control unit may be part of a larger master instrument.

In such an embodiment, a control unit is configured to detect if magnetic stir bar60is within reaction chamber50. A current necessary to run a motor serving as the source of motive power20without the presence of magnetic stir bar60is known. A control unit detects a difference in that current when magnetic stir bar60is present and moving within reaction chamber50. A lack of a change in current may represent an absence of magnetic stir bar60within reaction chamber50. A feedback loop provides input to magnetic mixing device10or a master instrument. An alert may be provided to a user regarding the absence of magnetic stir bar60and of any other potential problems with the magnetic mixing device.

In an alternative embodiment, a master device controls magnetic mixing device10as a slave device. In an exemplary embodiment, the master device is a master instrument that controls magnetic mixing device10.FIG. 4illustrates an exemplary logic of a master instrument in controlling a magnetic mixing device10. By way of background, when an assay is developed, an optimal thermal protocol, mixing protocol, and results interpretation methodology is prescribed. These pieces of information can be advantageously encoded onto an information carrier, else contained in a database that can be referenced by indicia. The information carrier can be in an illustrative embodiment a bar code, such as a 2-D barcode, which can contain, or provide the location of such information, for example a thermal protocol ID, a mixing protocol ID, a results interpretation ID, the manufacturing lot number of the assay, the catalog number of the assay, etc. At step130, the illustrative master instrument reads a bar code. At step140, the master instrument chooses a mixing protocol. This can be accomplished, for instance, by reference to an online or offline database and/or filesystem to retrieve the information referred to by the ID number. At step150, the master instrument sends commands to magnetic mixing device10. At step160, magnetic mixing device10sends a reply to the master instrument. In exemplary embodiments, the Instrument may communicate with the bar code module over USB, by wired or by wireless communications. The command structure in an exemplary embodiment may be modified as necessary to communicate with the bar code module employed. However any other communication protocol could be used, for instance serial, RS232, RS485, SPI, I2C, WiFi direct, Bluetooth, etc. Imaging systems such as cameras are also envisioned for capturing data labels, for example QR codes. RFID or other electromagnetic-based information tags can also be used to encode the information described above in place of an optical system.

Another embodiment of the present disclosure provides a kit for amplifying DNA from dried reaction components. The kit may comprise a magnetic mixing device as described above, dried down reaction components, and a thermocycler for DNA amplification.

An exemplary method of mixing by magnetic mixing device10will now be described. In describing the exemplary method, it will be assumed that a user is operating magnetic mixing device10shown inFIG. 1. However, it should be understood that an automated, semi-automated, or manually operated machine could also operate device10in a similar manner.

A user may fill reaction chamber50with the desired substances to be mixed, provided there is at least one substance that is a fluid. At least one magnetic stir bar60must be inserted into reaction chamber50, co-located with the substances to be mixed. Through movement of magnet40relative to a stationary reaction chamber50or movement of reaction chamber50relative to a stationary magnet40, magnetic stir bar60moves within reaction chamber50to create turbulent flow and uniform mixing throughout. Alternatively, a user may receive reaction chamber50containing at least one of the desired substances to be mixed, and/or magnetic stir bar60. Reaction chamber50may be contained within cartridge90. In an embodiment where magnetic stir bar60moves vertically throughout reaction chamber50, any air bubbles80present within or beneath the fluid are disrupted and/or moved to the top of reaction chamber50.

In an exemplary embodiment, a microprocessor may control the ramp rate and the speed of a motor serving as the source of motive power20.

In an exemplary embodiment where magnetic mixing device10is used for nucleic acid amplification, a user fills reaction chamber50with dried reaction components110and a liquid. Through the use of magnetic mixing device10, the dried reaction components110are rehydrated in the liquid. Alternatively, the user may receive reaction chamber50with dried reaction components110and magnetic stir bar60already within reaction chamber50. Reaction chamber50may be contained within cartridge90.

In an exemplary embodiment, magnetic mixing device10may be used before the reaction chamber is placed in a master instrument.

In an alternative embodiment, magnetic mixing device10may be used during the run on a master instrument. At a specified time, the user may remove the reaction chamber from the master instrument, place the reaction chamber in magnetic mixing device10, run a mixing protocol, and place the reaction chamber back in the master instrument.

In an alternative embodiment, one or more magnetic mixing devices may be incorporated as part of a master instrument, rather than as an accessory that communicates with a master instrument.

Table 1 presents data from a set of experiments where the magnetic mixing device was used for nucleic acid amplification. The data indicates the cycle threshold comparison between magnetic mixing, thermal mixing, and pipette mixing. The cycle threshold is the number of cycles of amplification required to cross a threshold value. The data shows magnetic mixing is equal to or better than conventional thermal mixing or pipette mixing, with the added advantages of being faster and allowing an unskilled user to operate the magnetic mixing device.

The foregoing description has been presented for purposes of illustration. It is not exhaustive and is not limited to the precise forms or embodiments disclosed. Exemplary embodiments have been presented as being used for nucleic acid amplification, this disclosure is not limited to nucleic acid amplification and can be used for mixing in any chemical or biological application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the embodiments disclosed herein.

Moreover, while illustrative embodiments have been described herein, the scope of any and all embodiments include equivalent elements, modifications, omissions, combinations (e.g., of aspects across various embodiments), adaptations and/or alterations as would be appreciated by those skilled in the art based on the present disclosure. The limitations in the claims are to be interpreted broadly based on the language employed in the claims and not limited to examples described in the present specification or during the prosecution of the application. The examples are to be construed as non-exclusive. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the embodiments being indicated by the following claims and their full scope of equivalents.